Selective antibody targeting of undifferentiated stem cells

ABSTRACT

This invention provides a system for producing differentiated cells from a stem cell population for use wherever a relatively homogenous cell population is desirable. The cells contain an effector gene under control of a transcriptional control element (such as the TERT promoter) that causes the gene to be expressed in relatively undifferentiated cells in the population. Expression of the effector gene results in expression of a cell-surface antigen that can be used to deplete the undifferentiated cells. Model effector sequences encode glycosyl transferases that synthesize carbohydrate xenoantigen or alloantigen, which can be used for immunoseparation or as a target for complement-mediated lysis. The differentiated cell populations produced are suitable for use in tissue regeneration and non-therapeutic applications such as drug screening.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. patent application 60/253,357;60/253,443; and 60/253,395, all filed Nov. 27, 2001, pending. Thepriority documents are hereby incorporated herein by reference in theirentirety.

TECHNICAL FIELD

This invention relates generally to the field of cell biology of stemcells, embryonic cells, and the molecular biology of promoter controlledviral vectors. More specifically, it describes a technology for removingundifferentiated cells from populations derived from pluripotent stemcells using selectively expressed lytic vectors.

BACKGROUND

Precursor cells have become a central interest in medical research. Manytissues in the body have a back-up reservoir of precursors that canreplace cells that are senescent or damaged by injury or disease.Considerable effort has been made recently to isolate precursors of anumber of different tissues for use in regenerative medicine.

U.S. Pat. No. 5,750,397 (Tsukamoto et al., Systemix) reports isolationand growth of human hematopoietic stem cells which are Thy-1+, CD34+,and capable of differentiation into lymphoid, erythroid, andmyelomonocytic lineages. U.S. Pat. No. 5,736,396 (Bruder et al.) reportsmethods for lineage-directed differentiation of isolated humanmesenchymal stem cells, using an appropriate bioactive factor. Thederived cells can then be introduced into a host for mesenchymal tissueregeneration or repair.

U.S. Pat. No. 5,716,411 (Orgill et al.) proposes regenerating skin atthe site of a burn or wound, using an epithelial autograft. U.S. Pat.No. 5,766,948 (F. Gage) reports a method for producing neuroblasts fromanimal brain tissue. U.S. Pat. No. 5,672,499 (Anderson et al.) reportsobtaining neural crest stem cells from embryonic tissue. U.S. Pat. No.5,851,832 (Weiss et al., Neurospheres) reports isolation of putativeneural stem cells from 8-12 week old human fetuses. U.S. Pat. No.5,968,829 (M. Carpenter) reports human neural stem cells derived fromprimary central nervous system tissue.

U.S. Pat. No. 5,082,670 (F. Gage) reports a method for graftinggenetically modified cells to treat defects, disease or damage of thecentral nervous system. Auerbach et al. (Eur. J. Neurosci. 12:1696,2000) report that multipotential CNS cells implanted into animal brainsform electrically active and functionally connected neurons. Brustle etal. (Science 285:754, 1999) report that precursor cells derived fromembryonic stem cells interact with host neurons and efficientlymyelinate axons in the brain and spinal cord.

Considerable interest has been generated by the development of embryonicstem cells, which are thought to have the potential to differentiateinto many cell types. Early work on embryonic stem cells was done inmice. Mouse stem cells can be isolated from both early embryonic cellsand germinal tissue. Desirable characteristics of pluripotent stem cellsare that they be capable of proliferation in vitro in anundifferentiated state, retain a normal karyotype, and retain thepotential to differentiate to derivatives of all three embryonic germlayers (endoderm, mesoderm, and ectoderm).

Development of human pluripotent stem cell preparations is considerablyless advanced than work with mouse cells. Thomson et al. propagatedpluripotent stem cells from lower primates (U.S. Pat. No. 5,843,780;Proc. Natl. Acad. Sci. USA 92:7844, 1995), and then from humans (Science282:114, 1998). Gearhart and coworkers derived human embryonic germ(hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc.Natl. Acad. Sci. USA 95:13726, 1998; and U.S. Pat. No. 6,090,622).

Both hES and hEG cells have the long-sought characteristics ofpluripotent stem cells: they are capable of being grown in vitro withoutdifferentiating, they have a normal karyotype, and they remain capableof producing a number of different cell types. Clonally derived humanembryonic stem cell lines maintain pluripotency and proliferativepotential for prolonged periods in culture (Amit et al., Dev. Biol.227:271, 2000). These cells hold considerable promise for use in humantherapy, acting as a reservoir for regeneration of almost any tissuecompromised by genetic abnormality, trauma, or a disease condition.

International Patent Publication WO 99/20741 (Geron Corp.) refers tomethods and materials for growing primate-derived primordial stem cells.In one embodiment, a cell culture medium is provided for growingprimate-derived primordial stem cells in a substantiallyundifferentiated state, having a low osmotic pressure and low endotoxinlevels. The basic medium is combined with a nutrient serum effective tosupport the growth of primate-derived primordial stem cells and asubstrate of feeder cells or an extracellular matrix component derivedfrom feeder cells. The medium can further include non-essential aminoacids, an anti-oxidant, and growth factors that are either nucleosidesor a pyruvate salt.

A significant challenge to the use of stem cells for therapy is tocontrol growth and differentiation into the particular type of tissuerequired for treatment of each patient.

U.S. Pat. No. 4,959,313 (M. Taketo, Jackson Labs) provides a particularenhancer sequence that causes expression of a flanking exogenous orrecombinant gene from a promoter accompanying the gene that does notnormally cause expression in undifferentiated cells. U.S. Pat. No.5,639,618 (D. A. Gay, Plurion Inc.) proposes a method for isolating alineage specific stem cell in vitro, in which a pluripotent embryonicstem cell is transfected with a construct in which a lineage-specificgenetic element is operably linked to a reporter gene, culturing thecell under conditions where the cell differentiates, and then separationof cells expressing the reporter are separated from other cells.

U.S. Pat. No. 6,087,168 (Levesque et. al., Cedars Sinai Med. Ctr.) isdirected to transdifferentiating epidermal cells into viable neuronsuseful for both cell therapy and gene therapy. Skin cells aretransfected with a neurogenic transcription factor, and cultured in amedium containing an antisense oligonucleotide corresponding to anegative regulator of neuronal differentiation.

International Patent Publication WO 97132025 (McIvor et al., U.Minnesota) proposes a method for engrafting drug-resistant hematopoieticstem cells. The cells in the graft are augmented by a drug resistancegene (such as methotrexate resistant dihydrofolate reductase), undercontrol of a promoter functional in stem cells. The cells areadministered into a mammal, which is then treated with the drug toincrease engraftment of transgenic cells relative to nontransgeniccells.

International Patent Publication WO 98/39427 (Stein et al., U.Massachusetts) refers to methods for expressing exogenous genes indifferentiated cells such as skeletal tissue. Stem cells (e.g., frombone marrow) are contacted with a nucleic acid in which the gene islinked to an element that controls expression in differentiated cells.Exemplary is the rat osteocalcin promoter. International PatentPublication WO 99/10535 (Liu et al., Yale U.) proposes a process forstudying changes in gene expression in stem cells. A gene expressionprofile of a stem cell population is prepared, and then compared a geneexpression profile of differentiated cells

International Patent Publication WO 99/19469 (Braetscher et al.,Biotransplant) refers to a method for growing pluripotent embryonic stemcells from the pig. A selectable marker gene is inserted into the cellsto be regulated by a control or promoter sequence in the ES cells,exemplified by the porcine OCT-4 promoter.

International Patent Publication WO 00/15764 (Smith et al., U.Edinburgh) refers to propagation and derivation of embryonic stem cells.The cells are cultured in the presence of a compound that selectivelyinhibits propagation or survival of cells other than ES cells byinhibiting a signaling pathway essential for the differentiated cells topropagate. Exemplary are compounds that inhibit SHP-2, MEK, or theras/MAPK cascade.

Klug et al. (J. Clin. Invest. 98:216, 1996) propose a strategy forgenetically selecting cardiomyocytes from differentiating mouseembryonic stem cells. A fusion gene consisting of the α-cardiac myosinheavy chain promoter and a cDNA encoding aminoglycosidephosphotransferase was stably transfected into the ES cells. Theresulting lines were differentiated in vitro and selected using G418.The selected cardiomyocyte cultures were reported to be highlydifferentiated. When engrafted back into mice, ES-derived cardiomyocytegrafts were detectable as long as 7 weeks after implantation.

Schuldiner et al. (Proc. Natl. Acad. Sci. USA 97:11307, 2000) report theeffects of eight growth factors on the differentiation of cells fromhuman embryonic stem cells. After initiating differentiation throughembryoid body formation, the cells were cultured in the presence ofbFGF, TGF-β1, activin-A, BMP-4, HGF, EGF, βNGF, or retinoic acid. Eachgrowth factor had a unique effect on the differentiation pathway, butnone of the growth factors directed differentiation exclusively to onecell type.

There is a need for new approaches to generate populations ofdifferentiated cells suitable for human administration.

SUMMARY OF THE INVENTION

This invention provides a system for depleting relativelyundifferentiated cells from a heterogeneous cell population, such as maybe obtained by differentiation of stem cells. The population is treatedwith a vector that puts an effector gene under control of a gene elementthat allows the gene to be expressed at a higher level in theundifferentiated subpopulation. Exemplary effector genes areglycosyltransferases, rendering undifferentiated cells separable usingspecific antibody, or susceptible to lysis by antibody plus complement.This produces a population relatively enriched for mature cells, andsuitable for use in regenerative medicine.

One embodiment of this invention is a method of producing differentiatedcells. A cell population comprising undifferentiated stem cells thatcontain a nucleic acid molecule comprising the structure P-X is treatedso as to cause at least some undifferentiated cells in the population todifferentiate. X is nucleic acid sequence that causes expression of acell surface antigen under control of transcriptional control element P,which has the effect of causing the surface antigen to be preferentiallyexpressed in undifferentiated cells. The connecting line in P-Xindicates that the genetic elements are operatively linked, whether ornot they are adjacent in the nucleic acid molecule.

After the cell population is differentiated, relatively undifferentiatedcells can be depleted from the population by combining the cells withligand specific for the antigen. In this context, the term “ligand”refers to any biological molecule (typically a protein) that binds theantigen with a specificity to discriminate the antigen from othermolecules on the cell surface or on other cells in the population.Suitable ligands include specific monoclonal or polyclonal antibody andlectins. Depletion can be effected, for example, by combining the cellswith ligand specific for the antigen, and separating cells that have notbound the ligand. Where the specific ligand is an antibody, it can becombined with the cells in culture, or the cells can be placed in asubject having circulating natural antibody, or antibody that has beeninduced by active or passive immunization. The undifferentiated cellscan be removed, for example, by an affinity separation technique, cellsorting, or by complement-mediated lysis in culture or in situ.

Another embodiment of the invention is a method for depletingundifferentiated stem cells from a cell population. Stem cells in thepopulation are genetically altered so that they contain a nucleic acidmolecule comprising the structure P-X as already described.Undifferentiated cells are then depleted from the population usingantibody specific for the cell surface antigen. The cell population canbe genetically altered when it is still predominantly undifferentiated(before being caused to differentiate), or when it already predominantlycomprises differentiated cells.

In certain embodiments, X encodes a transmembrane protein which itselfacts as the cell surface antigen. In other embodiments, X encodes anenzyme that in turn causes the antigen to be expressed on the cellsurface. Exemplary are glycosyltransferase enzymes, particularlyα(1,3)galactosyltransferase, which causes expression of the Galα(1,3)Galxenoantigen, and ABO blood group transferases, which cause expression ofthe ABO histo blood group alloantigens. In order to enhance dominance ofthe desired phenotype, the glycosyltransferase encoding region canincorporate a heterologous membrane anchoring segment or cytoplasmicdomain (either at the N- or C-terminus) that optimizes positioning ofthe enzyme within the Golgi apparatus.

In certain embodiments, P-X is an introduced heterologous molecule,meaning that the cell or its ancestors was genetically altered with avector comprising P-X. In other embodiments the cell or its ancestorswas genetically altered with a vector to place X under control of anendogenous transcriptional control element. Following transfection, Xcan be either transiently expressed in undifferentiated cells in thepopulation, or P-X can be inheritable and expressed in undifferentiatedprogeny. Non-limiting examples for P include the OCT-4 promoter, and thepromoter of telomerase reverse transcriptase (TERT). The cells can alsocontain a drug resistance gene Y under control of P.

A further embodiment of the invention is a stem cell genetically alteredto express a carbohydrate antigen no normally expressed by the cell,possibly an antigen recognized by a naturally occurring antibody. As anexample, the cell can be genetically altered with a glycosyltransferase, such as an α(1,3)galactosyltransferase, or an ABO bloodgroup transferase. Expression of the carbohydrate antigen can becontrolled by a transcriptional control element specific forundifferentiated cells.

The reagents and techniques of this invention can be brought to bear oncell populations containing many different types of stem cells, asdescribed below. They are especially suited for application to primatepluripotent stem cells, such as human embryonic stem cells.

Other embodiments of the invention will be apparent from the descriptionthat follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an analysis of OCT-4 and hTERT expression in hES cellscultured with feeder cells (mEF) or extracellular matrix (Matrigel® orlaminin) with regular medium (RM) or conditioned medium (CM). The upperpanel is a copy of a gel showing OCT-4 and hTERT expression at the mRNAlevel by RT-PCR. The lower panel is a bar graph comparing the level ofexpression for cells grown on different substrates, expressed as theratio of OCT-4 or hTERT to the 18 s standard. hES cells grown on Lamininand Matrigel® in conditioned medium have similar expression patterns tothose of cells grown on a feeder layer.

FIG. 2 is a half-tone reproduction of a gel showing telomerase activitymeasured in cultured hES cells by TRAP activity assay. All the cultureconditions showed positive telomerase activity after 40 days infeeder-free culture.

FIG. 3 is a half-tone reproduction showing expression of the GFPreporter gene in hES cells transduced with retrovirus and thendifferentiated. hES cells were transferred to suspension culture to formembryoid bodies, cultured for a further 4 days, replated ontogelatin-coated slides and cultured for a week, and then fixed andphotographed under fluorescence for GFP expression. Left panels showbright-field illumination; right panels show fluorescence due to GFPexpression.

FIG. 4 shows the results of a study in which hES cells were transientlygenetically altered in feeder-free culture by lipofection. Panel A is ahalf-tone reproduction of a light micrograph showing morphology of hEScells on laminin after they have been transfected. Panel B is ahalf-tone reproduction of a fluorescence micrograph showing GFPexpression in the same colony. Panel C is a bar graph showing percentageof cells expressing GFP under various conditions.

FIG. 5 is a map of TPAC vector designated pGRN376. This is an adenovirusvector of 7185 bp comprising the herpes simplex thymidine kinase (tk)gene under control of a promoter taken from the upstream sequence of thehuman gene for telomerase reverse transcriptase (hTERT). Expression oftk is promoted in cells expressing hTERT, such as undifferentiatedembryonic stem cells.

FIG. 6 is a two-panel line graph, showing the effect of the TPACthymidine kinase vector on undifferentiated hES cells. 48 h afterreplating, the cells were transduced with TPAC vector at an MOI of 30 or100, or mock transduced (no vector added). Four h later, the cells wereexchanged into fresh medium containing the prodrug ganciclovir (GCV). Byday 3, wells treated with TPAC vector+GCV contained 8% as many cells asthe control wells.

FIG. 7 is a bar graph showing titration of GCV in TPAC vector treatedhES cells. 4 h after transduction with the vector, fresh medium wasadded containing GCV at the concentration shown. ˜20 μM GCV was optimalunder the conditions tested.

FIG. 8 is a two-panel bar graph showing titration of GCV on TPAC vectortransduced and mock-transduced hES cells from two different lines. Bothlines are sensitive to GCV after treatment with the TPAC vector.

FIG. 9 shows the effect of TPAC+GCV treatment on mixed cell populationsobtained from differentiation of hES cells. The cells were fed dailywith conditioned medium to maintain the undifferentiated state, or witheither 500 nM retinoic acid or 0.5% DMSO, to induce differentiation intocommitted cells of mixed phenotype. 7 days later, they were infectedwith the TPAC vector at an MOI of 30, plus 20 μM GCV.

The Upper Panel is a bar graph showing the number of cells surviving inculture. Treatment with TPAC+GCV eliminated cells cultured under eachcondition. In each instance, culture of the surviving cells producedpopulations that appeared highly differentiated and substantially freeof undifferentiated morphology. The Lower Panel is a half-tonereproduction of a gel showing RT-PCR analysis of the surviving cells.Those cells cultured with conditioned medium (mEF-CM) or DMSO had nodetectable OCT-4 expression, while 2 out of 4 samples treated withretinoic acid (RA) showed amplification products consistent with verylow levels of OCT-4 expression.

FIG. 10 is a schematic depiction of targeting strategy to place anα(1,3)galactosyltransferase encoding sequence under control of theendogenous hTERT promoter on one allele (Example 13). In the two-stepapproach (upper panel), the endogenous hTERT gene is targeted with apromoterless vector comprising the neo gene, and selected for G418resistance. The neo sequence is then replaced withα(1,3)galactosyltransferase (α1,3GT) using cre recombinase. In the onestep approach (lower panel), neo is introduced with an internalribosomal entry site 3′ to the α1,3GT coding region. In this instance,the α1,3GT is truncated before the polyadenylation signal and istranscribed directly from the hTERT promoter. A bicistronic message isproduced from which both proteins are translated.

DETAILED DESCRIPTION OF THE INVENTION

Stem cells of various kinds have become an extremely attractive modalityin regenerative medicine. They can be proliferated in culture, and thendifferentiated in vitro or in situ into the cell types needed fortherapy. Recently, it has been demonstrated that human embryonic stemcells continuously express a high level of telomerase, enabling them tomaintain telomere length and grow almost indefinitely in culture.

So far, efforts to differentiate stem cells have been directed primarilytowards identifying culture conditions that promote outgrowth of a cellpopulation with phenotypic features of a tissue type desirable forregenerative medicine. Schuldiner et al. (supra) report the effects ofgrowth factors on the differentiation of human embryonic stem cells. InU.S. Pat. No. 5,639,613, stem cells are transfected with alineage-specific gene that is operably linked to a reporter gene, whichis then used to select for cells expressing the reporter. In WO97/32025, hematopoietic stem cells are augmented by a drug resistancegene, and then engrafted into a subject. The cells are administered intoa mammal, which is then treated with the drug to increase engraftment oftransgenic cells. Klug et al. (supra) used a construct in which theα-cardiac myosin heavy chain promoter controlled expression ofaminoglycoside phosphotransferase. Transfected differentiated cells wereselected using G418, which produced lines of cardiomyocyte like cells.This is a positive selection strategy that uses gene expression patternsof the desired tissue type to allow preferential survival ofdifferentiated tissue.

It is a hypothesis of this invention that positively selecting fordifferentiated cells produces populations that are suboptimal for use inhuman therapy. Any undifferentiated cells in the population may impairengraftment or function of the cells in vivo. Undifferentiated cells mayalso increase the possibility of a malignancy or other tumor forming atthe site of the therapeutic implant, or by migration of transplantedcells.

This invention is directed towards a strategy in which undifferentiatedcells remaining in a differentiated cell population are depleted. Thisis effected by genetically altering the cells, so that a gene that islethal to a cell in which it is expressed, or renders it susceptible toa lethal effect of an external agent, is placed under transcriptionalcontrol of a genetic element that causes it to be expressedpreferentially in any undifferentiated cells in the population. This isa negative selection strategy, designed to minimize the proportion ofundifferentiated cells. It is possible to combine this technique withpositive selection techniques of various kinds, in order to obtainrelatively pure populations of the desired tissue type that areessentially free of undifferentiated cells.

In certain embodiments of the invention, the cell population istransfected with a genetic construct in which a promoter specific forundifferentiated cells drives a glycosyl transferase, which in turnsynthesizes a new surface antigen. For example,α(1,3)galactosyltransferase (α1,3GT) can be used to express theGalα(1,3)Gal epitope on the cell surface. After culturing the cellsunder conditions where the antigen can be formed, the cells areseparated using specific antibody, or treated with specific antibody andcomplement to deplete undifferentiated cells from the population. Anumber of glycosyl transferases are suitable for this purpose,particularly those that synthesize a xenoantigen or alloantigen againstwhich humans have naturally occurring antibody.

To validate the negative selection strategy, human embryonic stem (hES)cells have been transduced with an adenovirus vector (TPAC) in which aherpes virus thymidine kinase gene was placed under control of apromoter sequence for human telomerase reverse transcriptase (hTERT).hES cells constitutively express hTERT, but this ability is lost upondifferentiation. Example 10 (FIGS. 6-8) show that transduction of hEScells with TPAC vector renders undifferentiated cells susceptible tolethality by the prodrug ganciclovir, a substrate for thymidine kinase,at a concentration of ˜20 μM. Example 11 (FIG. 9) shows that when hEScells are transduced with TPAC vector and then differentiated with DMSO,there are no surviving cells with detectable OCT-4 expression (aphenotype of undifferentiated cells).

The techniques of this invention are designed in part to provide cellpopulations with improved characteristics for human therapy. Afterdepleting undifferentiated cells, the differentiated population isexpected to possess better functional and engraftment characteristics,and have reduced risk of creating unwanted tissue architecture andmalignancies in the treated subject. In addition, cell populationsdepleted of undifferentiated cells are more homogeneous, which providesa distinct advantage for non-therapeutic applications, such as producingantibody, cDNA libraries, and screening drug candidates.

A particular advantage of using a glycosyl transferase as the effectorsequence is that the system provides ongoing surveillance after thecells are used for tissue regeneration. If undifferentiated cellsreappear in the transplanted tissue (either through dedifferentiation oroutgrowth of a preexisting subpopulation), the specific promoter willprompt synthesis of the glycosyltransferase—leading to expression of theantigen, followed by lysis of the undifferentiated cells in situ.

Another advantage is that the stem cells can be genetically altered withthe glycosyl transferase in advance, and passaged or stored until use.The stem cell line can then be differentiated when needed, and depletedof undifferentiated cells by separating or lysing cells expressing thecarbohydrate determinant synthesized by the transferase.

Definitions

Prototype “primate Pluripotent Stem cells” (pPS cells) are pluripotentcells derived from pre-embryonic, embryonic, or fetal tissue at any timeafter fertilization, and have the characteristic of being capable underappropriate conditions of producing progeny of several different celltypes that are derivatives of all of the three germinal layers(endoderm, mesoderm, and ectoderm), according to a standard art-acceptedtest, such as the ability to form a teratoma in 8-12 week old SCID mice.

Included in the definition of pPS cells are embryonic cells of varioustypes, exemplified by human embryonic stem (hES) cells, described byThomson et al. (Science 282:1145, 1998); embryonic stem cells from otherprimates, such as Rhesus stem cells (Thomson et al., Proc. Natl. Acad.Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol.Reprod. 55:254, 1996) and human embryonic germ (hEG) cells (Shamblott etal., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Other types ofpluripotent cells are also included in the term. Any cells of primateorigin that are capable of producing progeny that are derivatives of allthree germinal layers are included, regardless of whether they werederived from embryonic tissue, fetal tissue, or other sources. Thisinvention relates to pPS cells that are not derived from a malignantsource. It is desirable (but not always necessary) that the cells bekaryotypically normal.

pPS cell cultures are described as “undifferentiated” when a substantialproportion of stem cells and their derivatives in the population displaymorphological characteristics of undifferentiated cells, clearlydistinguishing them from differentiated cells of embryo or adult origin.Undifferentiated pPS cells are easily recognized by those skilled in theart, and typically appear in the two dimensions of a microscopic view incolonies of cells with high nuclear/cytoplasmic ratios and prominentnucleoli. It is understood that colonies of undifferentiated cellswithin the population will often be surrounded by neighboring cells thatare differentiated. Nevertheless, the undifferentiated colonies persistwhen the population is cultured or passaged under appropriateconditions, and individual undifferentiated cells constitute asubstantial proportion of the cell population. Cultures that aresubstantially undifferentiated contain at least 20% undifferentiated pPScells, and may contain at least 40%, 60%, or 80% in order of increasingpreference. Whenever a culture or cell population is referred to in thisdisclosure as proliferating “without differentiation”, what is meant isthat after proliferation, the composition is substantiallyundifferentiated according to the preceding definition.

“Feeder cells” or “feeders” are terms used to describe cells of one typethat are co-cultured with cells of another type, to provide anenvironment in which the cells of the second type can grow. The feedercells are optionally from a different species as the cells they aresupporting. For example, certain types of pPS cells can be supported byprimary mouse embryonic fibroblasts, immortalized mouse embryonicfibroblasts, or human fibroblast-like cells differentiated from hEScells, as described later in this disclosure. pPS cell populations aresaid to be “essentially free” of feeder cells if the cells have beengrown through at least one round after splitting in which fresh feedercells are not added to support the growth of the pPS. Culturesessentially free of feeder cells contain less than about 5% feedercells. Whenever a culture or cell population is referred to in thisdisclosure as “feeder-free”, what is meant is that the composition isessentially free of feeder cells according to the preceding definition,subject only to further constraints explicitly required.

The term “embryoid bodies” is a term of art synonymous with “aggregatebodies”. The terms refer to aggregates of differentiated andundifferentiated cells that appear when pPS cells overgrow in monolayercultures, or are maintained in suspension cultures. Embryoid bodies area mixture of different cell types, typically from several germ layers,distinguishable by morphological criteria.

The terms “committed precursor cells”, “lineage restricted precursorcells” and “restricted developmental lineage cells” all refer to cellsthat are capable of proliferating and differentiating into severaldifferent cell types, with a range that is typically more limited thanpluripotent stem cells of embryonic origin capable of giving rise toprogeny of all three germ layers. Non-limiting examples of committedprecursor cells include hematopoietic cells, which are pluripotent forvarious blood cells; hepatocyte progenitors, which are pluripotent forbile duct epithelial cells and hepatocytes; and mesenchymal stem cells.Another example is neural restricted cells, which can generate glialcell precursors that progress to oligodendrocytes and astrocytes, andneuronal precursors that progress to neurons.

For the purposes of this description, the term “stem cell” can refer toeither a pluripotent stem cell, or a committed precursor cell, both asdefined above. Minimally, a stem cell has the ability to proliferate andform cells of more than one different phenotype, and is also capable ofself renewal—either as part of the same culture, or when cultured underdifferent conditions. Embryonic stem cells can be identified as positivefor the enzyme telomerase.

As used in this disclosure, “differentiated” and “undifferentiated” arerelative terms depending on the context in which they are used.Specifically, in reference to a particular type of self-renewing stemcell, the term “undifferentiated” refers back to the same self-renewingstem cell, whereas the term “differentiated” refers to one or more ofthe relatively mature phenotypes the stem cell can generate—asdiscernable by morphological criteria, antigenic markers, and genetranscripts they produce. Undifferentiated pPS cells have the ability todifferentiate into all three germ layers. The cells differentiated fromthem do not, and can readily be recognized by one skilled in the art bymorphological criteria.

The terms “polynucleotide” and “nucleic acid molecule” refer to apolymer of nucleotides of any length. Included are genes and genefragments, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA and RNA, nucleic acid probes, and primers. As used in thisdisclosure, the term polynucleotides refer interchangeably to double-and single-stranded molecules. Unless otherwise specified or required,any embodiment of the invention that is a polynucleotide encompassesboth a double-stranded form, and each of the two complementarysingle-stranded forms known or predicted to make up the double-strandedform. Included are nucleic acid analogs such as phosporamidates andthiophosporamidates.

A cell is said to be “genetically altered”, “transfected”, or“genetically transformed” when a polynucleotide has been transferredinto the cell by any suitable means of artificial manipulation, or wherethe cell is a progeny of the originally altered cell that has inheritedthe polynucleotide. The polynucleotide will often comprise atranscribable sequence encoding a protein of interest, which enables thecell to express the protein at an elevated level. The genetic alterationis said to be “inheritable” if progeny of the altered cell have the samealteration.

A “control element” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules that contributes to thefunctional regulation of a polynucleotide, such as replication,duplication, transcription, splicing, translation, or degradation of thepolynucleotide. Transcriptional control elements include promoters,enhancers, and repressors.

Particular gene sequences referred to as promoters, like the “TERTpromoter”, or the “OCT-4 promoter”, are polynucleotide sequences derivedfrom the gene referred to that promote transcription of an operativelylinked gene expression product. It is recognized that various portionsof the upstream and intron untranslated gene sequence may in someinstances contribute to promoter activity, and that all or any subset ofthese portions may be present in the genetically engineered constructreferred to. The promoter may be based on the gene sequence of anyspecies having the gene, unless explicitly restricted, and mayincorporate any additions, substitutions or deletions desirable, as longas the ability to promote transcription in the target tissue. Geneticconstructs designed for treatment of humans typically comprise a segmentthat is at least 90% identical to a promoter sequence of a human gene. Aparticular sequence can be tested for activity and specificity, forexample, by operatively linking to a reporter gene (Example 9).

Genetic elements are said to be “operatively linked” if they are in astructural relationship permitting them to operate in a manner accordingto their expected function. For instance, if a promoter helps initiatetranscription of the coding sequence, the coding sequence can bereferred to as operatively linked to (or under control of) the promoter.There may be intervening sequence between the promoter and coding regionso long as this functional relationship is maintained.

In the context of encoding sequences, promoters, and other geneticelements, the term “heterologous” indicates that the element is derivedfrom a genotypically distinct entity from that of the rest of the entityto which it is being compared. For example, a promoter or geneintroduced by genetic engineering techniques into an animal of adifferent species is said to be a heterologous polynucleotide. An“endogenous” genetic element is an element that is in the same place inthe chromosome where it occurs in nature, although other elements may beartificially introduced into a neighboring position.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably in this disclosure to refer to polymers of amino acidsof any length. The polymer may comprise modified amino acids, it may belinear or branched, and it may be interrupted by non-amino acids.

The term “antibody” as used in this disclosure refers to both polyclonaland monoclonal antibody. The ambit of the term deliberately encompassesnot only intact immunoglobulin molecules, but also such fragments andgenetically engineered derivatives of immunoglobulin molecules as may beprepared by techniques known in the art, and which retains the bindingspecificity of the antigen binding site.

General Techniques

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, and embryology. Included areTeratocarcinomas and Embryonic Stem Cells: A Practical Approach (E. J.Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in MouseDevelopment (P. M. Wasserman et al., eds., Academic Press 1993);Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth.Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells:Prospects for Application to Human Biology and Gene Therapy (P. D.Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998). Differentiation ofstem cells is reviewed in Robertson, Meth. Cell Biol. 75:173, 1997; andPedersen, Reprod. Fertil. Dev. 10:31, 1998.

Methods in molecular genetics and genetic engineering are describedgenerally in the current editions of Molecular Cloning: A LaboratoryManual, (Sambrook et al.); Oligonucleotide Synthesis (M. J. Gait, ed.,);Animal Cell Culture (R. I. Freshney, ed.); Gene Transfer Vectors forMammalian Cells (Miller & Calos, eds.); Current Protocols in MolecularBiology and Short Protocols in Molecular Biology, 3rd Edition (F. M.Ausubel et al., eds.); and Recombinant DNA Methodology (R. Wu ed.,Academic Press). Reagents, cloning vectors, and kits for geneticmanipulation referred to in this disclosure are available fromcommercial vendors such as BioRad, Stratagene, Invitrogen, and ClonTech.

General techniques in cell culture and media collection are outlined inLarge Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol.8:148, 1997); Serum-free Media (K. Kitano, Biotechnology 17:73, 1991);Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2:375,1991); and Suspension Culture of Mammalian Cells (Birch et al.,Bioprocess Technol. 19:251, 1990).

Sources of Stem Cells

This invention can be practiced using stem cells of various types, whichmay include the following non-limiting examples.

U.S. Pat. No. 5,851,832 reports multipotent neural stem cells obtainedfrom brain tissue. U.S. Pat. No. 5,766,948 reports producing neuroblastsfrom newborn cerebral hemispheres. U.S. Pat. Nos. 5,654,183 and5,849,553 report the use of mammalian neural crest stem cells. U.S. Pat.No. 6,040,180 reports in vitro generation of differentiated neurons fromcultures of mammalian multipotential CNS stem cells. WO 98/50526 and WO99/01159 report generation and isolation of neuroepithelial stem cells,oligodendrocyte-astrocyte precursors, and lineage-restricted neuronalprecursors. U.S. Pat. No. 5,968,829 reports neural stem cells obtainedfrom embryonic forebrain and cultured with a medium comprising glucose,transferrin, insulin, selenium, progesterone, and several other growthfactors.

Primary liver cell cultures can be obtained from human biopsy orsurgically excised tissue by perfusion with an appropriate combinationof collagenase and hyaluronidase. Alternatively, EP 0 953 633 A1 reportsisolating liver cells by preparing minced human liver tissue,resuspending concentrated tissue cells in a growth medium and expandingthe cells in culture. The growth medium comprises glucose, insulin,transferrin, T₃, FCS, and various tissue extracts that allow thehepatocytes to grow without malignant transformation. The cells in theliver are thought to contain specialized cells including liverparenchymal cells, Kupffer cells, sinusoidal endothelium, and bile ductepithelium, and also precursor cells (referred to as “hepatoblasts” or“oval cells”) that have the capacity to differentiate into both maturehepatocytes or biliary epithelial cells (L. E. Rogler, Am. J. Pathol.150:591, 1997; M. Alison, Current Opin. Cell Biol. 10:710,1998; Lazaroet al., Cancer Res. 58:514, 1998).

U.S. Pat. No. 5,192,553 reports methods for isolating human neonatal orfetal hematopoietic stem or progenitor cells. U.S. Pat. No. 5,716,827reports human hematopoietic cells that are Thy-1 positive progenitors,and appropriate growth media to regenerate them in vitro. U.S. Pat. No.5,635,387 reports a method and device for culturing human hematopoieticcells and their precursors. U.S. Pat. No. 6,015,554 describes a methodof reconstituting human lymphoid and dendritic cells.

U.S. Pat. No. 5,486,359 reports homogeneous populations of humanmesenchymal stem cells that can differentiate into cells of more thanone connective tissue type, such as bone, cartilage, tendon, ligament,and dermis. They are obtained from bone marrow or periosteum. Alsoreported are culture conditions used to expand mesenchymal stem cells.WO 99/01145 reports human mesenchymal stem cells isolated fromperipheral blood of individuals treated with growth factors such asG-CSF or GM-CSF. WO 00/53795 reports adipose-derived stem cells andlattices, substantially free of adipocytes and red cells. These cellsreportedly can be expanded and cultured to produce hormones andconditioned culture media.

The invention can be practiced using stem cells of any vertebratespecies. Included are stem cells from humans; as well as non-humanprimates, domestic animals, livestock, and other non-human mammals.

Amongst the stem cells suitable for use in this invention are primatepluripotent stem (pPS) cells derived from tissue formed after gestation,such as a blastocyst, or fetal or embryonic tissue taken any time duringgestation. Non-limiting examples are primary cultures or establishedlines of embryonic stem cells.

Media and Feeder Cells

Media for isolating and propagating pPS cells can have any of severaldifferent formulas, as long as the cells obtained have the desiredcharacteristics, and can be propagated further. Suitable sources are asfollows: Dulbecco's modified Eagles medium (DMEM), Gibco # 11965-092;Knockout Dulbecco's modified Eagles medium (KO DMEM), Gibco # 10829-018;200 mM L-glutamine, Gibco # 15039-027; non-essential amino acidsolution, Gibco 11140-050; β-mercaptoethanol, Sigma # M7522; humanrecombinant basic fibroblast growth factor (bFGF), Gibco # 13256-029.Exemplary serum-containing ES medium is made with 80% DMEM (typically KODMEM), 20% defined fetal bovine serum (FBS) not heat inactivated, 0.1 mMnon-essential amino acids, 1 mM L-glutamine, and 0.1 mMβ-mercaptoethanol. The medium is filtered and stored at 4° C. for nolonger than 2 weeks. Serum-free ES medium is made with 80% KO DMEM, 20%serum replacement, 0.1 mM non-essential amino acids, 1 mM L-glutamine,and 0.1 mM β-mercaptoethanol. An effective serum replacement is Gibco #10828-028. The medium is filtered and stored at 4° C. for no longer than2 weeks. Just before use, human bFGF is added to a final concentrationof 4 ng/mL (Bodnar et al., Geron Corp, International Patent PublicationWO 99/20741).

Feeder cells (where used) are propagated in mEF medium, containing 90%DMEM (Gibco # 11965-092), 10% FBS (Hyclone # 30071-03), and 2 mMglutamine. mEFs are propagated in T150 flasks (Corning #430825),splitting the cells 1:2 every other day with trypsin, keeping the cellssubconfluent. To prepare the feeder cell layer, cells are irradiated ata dose to inhibit proliferation but permit synthesis of importantfactors that support hES cells (˜4000 rads gamma irradiation). Six-wellculture plates (such as Falcon # 304) are coated by incubation at 37° C.with 1 mL 0.5% gelatin per well overnight, and plated with 375,000irradiated mEFs per well. Feeder cell layers are typically used 5 h to 4days after plating. The medium is replaced with fresh hES medium justbefore seeding pPS cells.

Conditions for culturing other stem cells are known, and can beoptimized appropriately according to the cell type. Media and culturetechniques for particular cell types referred to in the previous sectionare provided in the references cited.

Embryonic Stem Cells

Embryonic stem cells can be isolated from blastocysts of members of theprimate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844,1995). Human embryonic stem (hES) cells can be prepared from humanblastocyst cells using the techniques described by Thomson et al. (U.S.Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133ff., 1998) and Reubinoff et al, Nature Biotech. 18:399, 2000.

Briefly, human blastocysts are obtained from human in vivopreimplantation embryos. Alternatively, in vitro fertilized (IVF)embryos can be used, or one cell human embryos can be expanded to theblastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Human embryosare cultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner etal., Fertil. Steril. 69:84, 1998). Blastocysts that develop are selectedfor ES cell isolation. The zona pellucida is removed from blastocysts bybrief exposure to pronase (Sigma). The inner cell masses are isolated byimmunosurgery, in which blastocysts are exposed to a 1:50 dilution ofrabbit anti-human spleen cell antiserum for 30 minutes, then washed for5 minutes three times in DMEM, and exposed to a 1:5 dilution of Guineapig complement (Gibco) for 3 minutes (see Solter et al., Proc. Natl.Acad. Sci. USA 72:5099, 1975). After two further washes in DMEM, lysedtrophectoderm cells are removed from the intact inner cell mass (ICM) bygentle pipetting, and the ICM plated on mEF feeder layers.

After 9 to 15 days, inner cell mass-derived outgrowths are dissociatedinto clumps either by exposure to calcium and magnesium-freephosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispaseor trypsin, or by mechanical dissociation with a micropipette; and thenreplated on mEF in fresh medium. Dissociated cells are replated on mEFfeeder layers in fresh ES medium, and observed for colony formation.Colonies demonstrating undifferentiated morphology are individuallyselected by micropipette, mechanically dissociated into clumps, andreplated. ES-like morphology is characterized as compact colonies withapparently high nucleus to cytoplasm ratio and prominent nucleoli.Resulting ES cells are then routinely split every 1-2 weeks by brieftrypsinization, exposure to Dulbecco's PBS (without calcium or magnesiumand with 2 mM EDTA), exposure to type IV collagenase (˜200 U/mL; Gibco)or by selection of individual colonies by micropipette. Clump sizes ofabout 50 to 100 cells are optimal.

Embryonic Germ Cells

Human Embryonic Germ (hEG) cells can be prepared from primordial germcells present in human fetal material taken about 8-11 weeks after thelast menstrual period. Suitable preparation methods are described inShamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S.Pat. No. 6,090,622.

Briefly, genital ridges are rinsed with isotonic buffer, then placedinto 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution (BRL) and cutinto <1 mm³ chunks. The tissue is then pipetted through a 100 μL tip tofurther disaggregate the cells. It is incubated at 37° C. for ˜5 min,then ˜3.5 mL EG growth medium is added. EG growth medium is DMEM, 4500mg/L D-glucose, 2200 mg/L mM sodium bicarbonate; 15% ES qualified fetalcalf serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL);1000-2000 U/mL human recombinant leukemia inhibitory factor (LIF,Genzyme); 1-2 ng/ml human recombinant basic fibroblast growth factor(bFGF, Genzyme); and 10 μM forskolin (in 10% DMSO). In an alternativeapproach, EG cells are isolated using hyaluronidase/collagenase/DNAse.Gonadal anlagen or genital ridges with mesenteries are dissected fromfetal material, the genital ridges are rinsed in PBS, then placed in 0.1mL HCD digestion solution (0.01% hyaluronidase type V, 0.002% DNAse I,0.1% collagenase type IV, all from Sigma prepared in EG growth medium).Tissue is minced and incubated 1 h or overnight at 37° C., resuspendedin 1-3 mL of EG growth medium, and plated onto a feeder layer.

Ninety-six well tissue culture plates are prepared with a sub-confluentlayer of feeder cells cultured for 3 days in modified EG growth mediumfree of LIF, bFGF or forskolin, inactivated with 5000 rad γ-irradiation.Suitable feeders are STO cells (ATCC Accession No. CRL 1503). ˜0.2 mL ofprimary germ cell (PGC) suspension is added to each of the wells. Thefirst passage is conducted after 7-10 days in EG growth medium,transferring each well to one well of a 24-well culture dish previouslyprepared with irradiated STO mouse fibroblasts. The cells are culturedwith daily replacement of medium until cell morphology consistent withEG cells are observed, typically after 7-30 days or 1-4 passages.

Propagation of pPS Cells in an Undifferentiated State

pPS cells can be propagated continuously in culture, using a combinationof culture conditions that promote proliferation without promotingdifferentiation.

Traditionally, pPS cells are cultured on a layer of feeder cells,typically fibroblast type cells, often derived from embryonic or fetaltissue. The cell lines are plated to near confluence, usually irradiatedto prevent proliferation, and then used to support pPS cell cultures.

In one illustration, pPS cells are first derived and supported onprimary embryonic fibroblasts. Mouse embryonic fibroblasts (mEF) can beobtained from outbred CF1 mice (SASCO) or other suitable strains. Theabdomen of a mouse at 13 days of pregnancy is swabbed with 70% ethanol,and the decidua is removed into phosphate buffered saline (PBS). Embryosare harvested; placenta, membranes, and soft tissues are removed; andthe carcasses are washed twice in PBS. They are then transferred tofresh 10 cm bacterial dishes containing 2 mL trypsin/EDTA, and finelyminced. After incubating 5 min at 37° C., the trypsin is inactivatedwith 5 mL DMEM containing 10% bovine serum (FBS), and the mixture istransferred to a 15 mL conical tube and dissociated. Debris is allowedto settle for 2 min, the supernatant is made up to a final volume of 10mL, and plated onto a 10 cm tissue culture plate or T75 flask. The flaskis incubated undisturbed for 24 h, after which the medium is replaced.When flasks are confluent (˜2-3 d), they are split 1:2 into new flasks.

Scientists at Geron have discovered that hPS cells can be maintained inan undifferentiated state even without feeder cells. The environment forfeeder-free cultures includes a suitable culture substrate, particularlyan extracellular matrix, such as may be derived from basement membraneor that may form part of adhesion molecule receptor-ligand couplings. Asuitable preparation available from Becton Dickenson under the nameMatrigel®. Other extracellular matrix components and component mixturesare suitable as an alternative. Depending on the cell type beingproliferated, this may include laminin, fibronectin, proteoglycan,entactin, heparan sulfate, and the like, alone or in variouscombinations. Laminins are major components of all basal laminae invertebrates, which interact with integrin heterodimers such as α6β1 andα6β4 (specific for laminins) and other heterodimers (that cross-reactwith other matrices).

The pluripotent stem cells are plated onto the substrate in a suitabledistribution and in the presence of a medium that promotes cellsurvival, propagation, and retention of the desirable characteristics.It has been found that plating densities of at least ˜15,000 cells cm⁻²(typically 90,000 cm⁻² to 170,000 cm⁻²) promote survival and limitdifferentiation. The passage of pPS cells in the absence of feedersbenefits from preparing the pPS cells in small clusters. Typically,enzymatic digestion is halted before cells become completely dispersed(say, ˜5 min with collagenase IV). Clumps of ˜10-2000 cells are thenplated directly onto the substrate without further dispersal.

Alternatively, primate PS cells can be passaged between feeder-freecultures as a finer cell suspension, providing that an appropriateenzyme and medium are chosen, and the plating density is sufficientlyhigh. By way of illustration, confluent human embryonic stem cellscultured in the absence of feeders are removed from the plates byincubating with a solution of 0.05% (wt/vol) trypsin (Gibco) and 0.053mM EDTA for 5-15 min at 37° C. With the use of pipette, the remainingcells in the plate are removed and the cells are triturated with thepipette until the cells are dispersed into a suspension comprisingsingle cells and some small clusters. The cells are then plated atdensities of 50,000-200,000 cells/cm² to promote survival and limitdifferentiation. The phenotype of ES cells passaged by this technique issimilar to what is observed when cells are harvested as clusters bycollagen digestion. As another option, the cells can be harvestedwithout enzymes before the plate reaches confluence. The cells areincubated ˜5 min in a solution of 0.5 mM EDTA alone in PBS, washed fromthe culture vessel, and then plated into a new culture without furtherdispersal.

pPS cells plated in the absence of fresh feeder cells benefit from beingcultured in a nutrient medium. The medium will generally contain theusual components to enhance cell survival, including isotonic buffer,essential minerals, and either serum or a serum replacement of somekind. Conditioned medium can be prepared by culturing irradiated primarymouse embryonic fibroblasts (or another suitable cell preparation) at adensity of ˜5-6×10⁴ cm⁻² in a serum free medium such as KO DMEMsupplemented with 20% serum replacement and 4 ng/mL basic fibroblastgrowth factor (bFGF). The culture supernatant is harvested after ˜1 dayat 37° C.

As an alternative to primary mouse fibroblast cultures, conditionedmedium can be prepared from an embryonic fibroblast cell line tested forits ability to condition medium appropriately. Such lines can optionallybe transfected with telomerase reverse transcriptase to increase theirreplicative capacity. Another possible source is differentiated pPScells with the morphological features of fibroblasts. pPS cells aresuspension cultured as aggregates in differentiation medium usingnon-adherent cell culture plates (˜2×10⁶ cells/9.6 cm²). After 2 daysthe aggregates are transferred into gelatin-coated plates, andfibroblast-like cells appear in clusters of 100-1000 cells in the mixedpopulation after ˜11 days. After brief collagenase treatment, thefibroblast-like cells can be collected under a microscope, passaged inmEF medium, and tested for their ability to condition ES medium.

Medium that has been conditioned for 1-2 days is typically used tosupport pPS cell culture for 1-2 days, and then exchanged. If desired,conditioned medium can be supplemented before use with additional growthfactors that benefit pPS cell culture. For hES, a growth factor likebFGF or FGF-4 can be used. For hEG, culture medium may be supplementedwith a growth factor like bFGF, an inducer of gp130, such as LIF orOncostatin-M, and perhaps a factor that elevates cyclic AMP levels, suchas forskolin.

Characteristics of Undifferentiated pPS Cells

In the two dimensions of a standard microscopic image, hES cells havehigh nuclear/cytoplasmic ratios in the plane of the image, prominentnucleoli, and compact colony formation with poorly discernable celljunctions. Cell lines can be karyotyped using a standard G-bandingtechnique (available at many clinical diagnostics labs that providesroutine karyotyping services, such as the Cytogenetics Lab at OaklandCalif.) and compared to published human karyotypes.

hES and hEG cells can also be characterized on the basis of expressedcell markers. In general, the tissue-specific markers discussed in thisdisclosure can be detected using a suitable immunological technique—suchas flow cytometry for membrane-bound markers, immunohistochemistry forintracellular markers, and enzyme-linked immunoassay, for markerssecreted into the medium. The expression of protein markers can also bedetected at the mRNA level by reverse transcriptase-PCR usingmarker-specific primers. See U.S. Pat. No. 5,843,780 for furtherdetails.

Stage-specific embryonic antigens (SSEA) are characteristic of certainembryonic cell types. Antibodies for SSEA markers are available from theDevelopmental Studies Hybridoma Bank (Bethesda Md.). Other usefulmarkers are detectable using antibodies designated Tra-1-60 and Tra-1-81(Andrews et al., Cell Lines from Human Germ Cell Tumors, in E. J.Robertson, 1987, supra). hES cells are typically SSEA-1 negative andSSEA-4 positive. hEG cells are typically SSEA-1 positive.Differentiation of pPS cells in vitro results in the loss of SSEA-4,Tra-1-60, and Tra-1-81 expression and increased expression of SSEA-1.pPS cells can also be characterized by the presence of alkalinephosphatase activity, which can be detected by fixing the cells with 4%paraformaldehyde, and then developing with Vector Red as a substrate, asdescribed by the manufacturer (Vector Laboratories, Burlingame Calif.).

Embryonic stem cells are also typically telomerase positive and OCT-4positive. Telomerase activity can be determined using TRAP activityassay (Kim et al., Science 266:2011, 1997), using a commerciallyavailable kit (TRAPeze® XK Telomerase Detection Kit, Cat. s7707;Intergen Co., Purchase N.Y.; or TeloTAGGG™ Telomerase PCR ELISAplus,Cat. 2,013,89; Roche Diagnostics, Indianapolis). hTERT expression canalso be evaluated at the mRNA level by RT-PCR. The LightCyclerTeloTAGGG™ hTERT quantification kit (Cat. 3,012,344; Roche Diagnostics)is available commercially for research purposes.

Differentiating pPS Cells

Differentiation of the pPS can be initiated by first forming embryoidbodies. General principles in culturing embryoid bodies are reported inO'Shea, Anat. Rec. (New Anat. 257:323, 1999). pPS cells are cultured ina manner that permits aggregates to form, for which many options areavailable: for example, by overgrowth of a donor pPS cell culture, or byculturing pPS cells in culture vessels having a substrate with lowadhesion properties which allows EB formation. Embryoid bodies can alsobe made in suspension culture. pPS cells are harvested by briefcollagenase digestion, dissociated into clusters, and plated innon-adherent cell culture plates. The aggregates are fed every few days,and then harvested after a suitable period, typically 4-8 days. Thecells can then be cultured in a medium and/or on a substrate thatpromotes enrichment of cells of a particular lineage. The substrate cancomprise matrix components such as Matrigel® (Becton Dickenson),laminin, collagen, gelatin, or matrix produced by first culturing amatrix-producing cell line (such as a fibroblast or endothelial cellline), and then lysing and washing in such a way that the matrix remainsattached to the surface of the vessel. Embryoid bodies comprise aheterogeneous cell population, potentially having an endoderm exterior,and a mesoderm and ectoderm interior.

Scientists at Geron Corporation have discovered that pPS cells can bedifferentiated into committed precursor cells or terminallydifferentiated cells without forming embryoid bodies or aggregates as anintermediate step. Briefly, a suspension of undifferentiated pPS cellsis prepared, and then plated onto a solid surface that promotesdifferentiation. Suitable substrates include glass or plastic surfacesthat are adherent. For example, glass coverslips can be coated with apolycationic substance, such as a polyamines like poly-lysine,poly-ornithine, or other homogeneous or mixed polypeptides or otherpolymers with a predominant positive charge. The cells are then culturedin a suitable nutrient medium that is adapted to promote differentiationtowards the desired cell lineage.

In some circumstances, differentiation is further promoted bywithdrawing serum or serum replacement from the culture medium. This canbe achieved by substituting a medium devoid of serum and serumreplacement, for example, at the time of replating. In certainembodiments of the invention, differentiation is promoted by withdrawingone or more medium component(s) that promote(s) growth ofundifferentiated cells, or act(s) as an inhibitor of differentiation.Examples of such components include certain growth factors, mitogens,leukocyte inhibitory factor (LIF), and basic fibroblast growth factor(bFGF). Differentiation may also be promoted by adding a mediumcomponent that promotes differentiation towards the desired celllineage, or inhibits the growth of cells with undesired characteristics.For example, to generate cells committed to neural or glial lineages,the medium can include any of the following factors or mediumconstituents in an effective combination: Brain derived neurotrophicfactor (BDNF), neutrotrophin-3 (NT-3), NT-4, epidermal growth factor(EGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF),retinoic acid (RA), sonic hedgehog, FGF-8, ascorbic acid, forskolin,fetal bovine serum (FBS), and bone morphogenic proteins (BMPs).

General principals for obtaining tissue cells from pluripotent stemcells are reviewed in Pedersen (Reprod. Fertil. Dev. 6:543, 1994), andU.S. Pat. No. 6,090,622. Other publications of interest include thefollowing: For neural progenitors, neural restrictive cells and glialcell precursors, see Bain et al., Biochem. Biophys. Res. Commun.200:1252, 1994; Trojanowski et al., Exp. Neurol. 144:92, 1997; Wojcik etal., Proc. Natl. Acad. Sci. USA 90:1305-130; and U.S. Pat. Nos.5,851,832, 5,928,947, 5,766,948, and 5,849,553. For cardiac muscle andcardiomyocytes see Chen et al., Dev. Dynamics 197:217, 1993 and Wobus etal., Differentiation 48:173, 1991. For hematopoietic progenitors, seeBurkert et al., New Biol. 3:698, 1991 and Biesecker et al., Exp.Hematol. 21:774, 1993. U.S. Pat. No. 5,773,255 relates toglucose-responsive insulin secreting pancreatic beta cell lines. U.S.Pat. No. 5,789,246 relates to hepatocyte precursor cells. Otherprogenitors of interest include but are not limited to chondrocytes,osteoblasts, retinal pigment epithelial cells, fibroblasts, skin cellssuch as keratinocytes, dendritic cells, hair follicle cells, renal ductepithelial cells, smooth and skeletal muscle cells, testicularprogenitors, and vascular endothelial cells.

Scientists at Geron Corporation have discovered that culturing pPS cellsor embryoid body cells in the presence of ligands that bind growthfactor receptors promotes enrichment for neural precursor cells. Thegrowth environment may contain a neural cell supportive extracellularmatrix, such as fibronectin. Suitable growth factors include but are notlimited to EGF, bFGF, PDGF, IGF-1, and antibodies to receptors for theseligands. The cultured cells may then be optionally separated on thebasis of whether they express a marker such as A2B5. Under theappropriate circumstances, populations of cells enriched for expressionof the A2B5 marker may have the capacity to generate both neuronal cells(including mature neurons), and glial cells (including astrocytes andoligodendrocytes). Optionally, the cell populations are furtherdifferentiated, for example, by culturing in a medium containing anactivator of cAMP.

Scientists at Geron Corporation have also discovered that culturing pPScells or embryoid body cells in the presence of a hepatocytedifferentiation agent promotes enrichment for hepatocyte-like cells. Thegrowth environment may contain a hepatocyte supportive extracellularmatrix, such as collagen or Matrigel®. Suitable differentiation agentsinclude various isomers of butyrate and their analogs, exemplified byn-butyrate. The cultured cells are optionally cultured simultaneously orsequentially with a hepatocyte maturation factor, such as an organicsolvent like dimethyl sulfoxide (DMSO); a maturation cofactor such asretinoic acid; or a cytokine or hormone such as a glucocorticoid,epidermal growth factor (EGF), insulin, TGF-α, TGF-β, fibroblast growthfactor (FGF), heparin, hepatocyte growth factor (HGF), IL-1, IL-6,IGF-I, IGF-II, and HBGF-1.

Characteristics of Differentiated Cells

Cells can be characterized according to a number of phenotypic criteria.The criteria include but are not limited to characterization ofmorphological features, detection or quantitation of expressed cellmarkers and enzymatic activity, and determination of the functionalproperties of the cells in vivo.

Markers of interest for neural cells include β-tubulin III orneurofilament, characteristic of neurons; glial fibrillary acidicprotein (GFAP), present in astrocytes; galactocerebroside (GalC) ormyelin basic protein (MBP); characteristic of oligodendrocytes; OCT-4,characteristic of undifferentiated hES cells; nestin, characteristic ofneural precursors and other cells. A2B5 and NCAM are characteristic ofglial progenitors and neural progenitors, respectively. Cells can alsobe tested for secretion of characteristic biologically activesubstances. For example, GABA-secreting neurons can be identified byproduction of glutamic acid decarboxylase or GABA. Dopaminergic neuronscan be identified by production of dopa decarboxylase, dopamine, ortyrosine hydroxylase.

Markers of interest for liver cells include α-fetoprotein (liverprogenitors); albumin, α₁-antitrypsin, glucose-6-phosphatase, cytochromep450 activity, transferrin, asialoglycoprotein receptor, and glycogenstorage (hepatocytes); CK7, CK19, and γ-glutamyl transferase (bileepithelium). It has been reported that hepatocyte differentiationrequires the transcription factor HNF-4α (Li et al., Genes Dev. 14:464,2000). Markers independent of HNF-4α expression include α₁-antitrypsin,α-fetoprotein, apoE, glucokinase, insulin growth factors 1 and 2, IGF-1receptor, insulin receptor, and leptin. Markers dependent on HNF-4αexpression include albumin, apoAI, apoAII, apoB, apoCIII, apoCII,aldolase B, phenylalanine hydroxylase, L-type fatty acid bindingprotein, transferrin, retinol binding protein, and erythropoietin (EPO).

Cell types in mixed cell populations derived from pPS cells can berecognized by characteristic morphology and the markers they express.For skeletal muscle: myoD, myogenin, and myf-5. For endothelial cells:PECAM (platelet endothelial cell adhesion molecule), Flk-1, tie-1,tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Forsmooth muscle cells: specific myosin heavy chain. For cardiomyocytes:GATA-4, Nkx2.5, cardiac troponin I, α-myosin heavy chain, and ANF. Forpancreatic cells, pdx and insulin secretion. For hematopoietic cells andtheir progenitors: GATA-1, CD34,AC133, β-major globulin, and β-majorglobulin like gene βH1.

Certain tissue-specifIc markers listed in this disclosure or known inthe art be detected by immunological techniques—such as flowimmunocytochemistry for cell-surface markers, immunohistochemistry (forexample, of fixed cells or tissue sections) for intracellular orcell-surface markers, Western blot analysis of cellular extracts, andenzyme-linked immunoassay, for cellular extracts or products secretedinto the medium. The expression of tissue-specific gene products canalso be detected at the mRNA level by Northern blot analysis, dot-blothybridization analysis. or by reverse transcriptase initiated polymerasechain reaction (RT-PCR) using sequence-specific primers in standardamplification methods. Sequence date for the particular markers listedin this disclosure can be obtained from public databases such as GenBankwhich is accessible over the internet.

Preparing Cell Populations Essentially Free of Undifferentiated Cells

In accordance with this invention, populations of differentiated cellsare depleted of relatively undifferentiated cells by expressing a genethat is lethal to cells or renders them susceptible to a lethal effectof an external agent, under control of a transcriptional control elementthat causes the gene to be preferentially expressed in theundifferentiated cells.

To accomplish this, the cells are genetically altered either before orafter the process used to differentiate the cells into the desiredlineage for therapy, in a way that puts an effector gene suitable fornegative selection of undifferentiated cells, under control of atranscriptional control element with the desired properties.

Transcriptional Control Elements for Driving Negative Selection

The control element is selected with a view to the protein expressionpatterns of the undifferentiated and differentiated cells in thepopulation.

Genes with desirable expression patterns can be identified by comparingexpression at the transcription, translation, or functional level in twodifferent cell populations—one relatively enriched for differentiatedcells, the other relatively enriched for undifferentiated cells.Suitable methods of comparison include subtractive hybridization of cDNAlibraries, and microarray analysis of mRNA levels. Once a transcript isidentified with an appropriate expression pattern, the promoter orenhancer of the corresponding gene can be used for construction of thenegative selection vector.

A suitable microarray analysis is conducted using a Genetic Microsystemsarray generator, and an Axon GenePix™ Scanner. Microarrays are preparedby amplifying cDNA fragments in a 96 or 384 well format, and thenspotted directly onto glass slides. To compare mRNA preparations fromtwo cell populations, one preparation is converted into Cy3-labeledcDNA, while the other is converted into Cy5-labeled cDNA. The two cDNApreparations are hybridized simultaneously to the microarray slide, andthen washed to eliminate non-specific binding. Any given spot on thearray will bind each of the EDNA products in proportion to abundance ofthe transcript in the two original mRNA preparations. The slide is thenscanned at wavelengths appropriate for each of the labels, and therelative abundance of mRNA is determined. Preferably, the level ofexpression of the effector gene will be at least 5-fold or even 25-foldhigher in the undifferentiated cells relative to the differentiatedcells.

For the depletion of pluripotent embryonic cells, an exemplary controlelement is the promoter for telomerase reverse transcriptase (TERT).Sequence of the human TERT gene (including upstream promoter sequence)is provided below. The reader is also referred to U.K. Patent GB 2321642B (Cech et al., Geron Corporation and U. Colorado), International PatentPublications WO 00/46355 (Morin et al., Geron Corporation), WO 99/33998(Hagen et al., Bayer Aktiengesellschaft), and Horikawa, I., et al.(Cancer Res., 59:826, 1999). Sequence of the mouse TERT gene is providedin WO 99/27113 (Morin et al., Geron Corporation). A lambda phage clonedesignated λGΦ5, containing ˜13,500 bases upstream from the hTERTencoding sequence is available from the ATCC under Accession No. 98505.Example 9 illustrates the testing and use of TERT promoter sequences(SEQ. ID NO:1) in vector expression systems.

Another exemplary control element is a promoter sequence for Octamerbinding transcription factor 4 (OCT-4), a member of the POU family oftranscription factors. OCT-4 transcription is activated between the 4and 80 cell stage in the developing embryo, and it is highly expressedin the expanding blastocyst and then in the pluripotent cells of the eggcylinder. Transcription is down-regulated as the primitive ectodermdifferentiates to form mesoderm, and by 8.5 days post coitum isrestricted to migrating primordial germ cells. High-level OCT-4 geneexpression is also observed in pluripotent embryo carcinoma andembryonic stem cell lines, and is down-regulated when these cells areinduced to differentiate. Pig, mouse, and human OCT-4 promoter sequencesare provided in International Patent Publication WO 9919469(Biotransplant Inc.).

Other suitable control elements can be obtained from genes causingexpression of markers characteristic of undifferentiated cells in thepopulation but not of the differentiated cells. For example, SSEA-3,SSEA-4, Tra-1-60 and Tra-1-81 are characteristic of various types ofundifferentiated pluripotent embryonic stem cells. The enzymeresponsible for synthesis of SSEA-4 may have transcriptional controlelements with the desirable expression specificity. A more recentexample is the promoter for Rex1 protein, a retanoic acid regulated zincfinger protein that is expressed in preimplantation embryos. The mouseRex1 promoter has been shown to act as an effective transcription markerfor undifferentiated embryonic stem cells (Eiges et al., Current Biol.11:514, 2001.

Suitability of particular elements can be estimated by analysis of genetranscript expression, for example, by microarray analysis. Reporterconstructs can then be tested in differentiated and undifferentiatedcells for the appropriate specificity, using a promoter or enhancersequence from the identified cell-specific gene to control transcriptionof a reporter gene, such as green fluorescence protein, secretedalkaline phosphatase, β-glucuronidase, or β-galactosidase. Use ofreporter constructs to test promoter specificity is illustrated below inExample 9.

Effector Genes for Achieving Negative Selection

A transcriptional regulatory element with appropriate specificity isoperatively linked to an encoding region for a product that will provideelimination of cells in which it is expressed—either directly, or byrendering the cell susceptible to an otherwise innocuous external agent.

Of particular interest are genes that cause presentation of a foreignantigen on the cell membrane. The presented substance may be analloantigen, a xenoantigen, or an antigen from a non-mammalian speciesfor which specific antibody is readily available. Expression of the geneleads to presentation of the antigen on undifferentiated cells, whichthen can be used to effect depletion by a suitable immunologicalseparation.

In one embodiment, the effector sequence encodes a membrane protein thatcontains the epitope recognized by the specific antibody. The membraneprotein may be a protein expressed in the same species on other types ofcells, but more typically is obtained from another species, or is anartificial sequence. In this case, the antigen will be foreign to thespecies from which the stem cells are derived, and antibodies made inthe same species will not cross-react with other antigens on the cell.Included are xenoantigens, alloantigens, and artificial antigens (madeby constructing an immunogenic peptide sequence not encoded in the humangenome).

In another embodiment, the target antigen is not a protein, but acarbohydrate or lipid component. In this case, the effector sequencewill encode an enzyme involved in antigen synthesis. Of particularinterest are glycosyl transferases of mammalian or non-mammalian originthat synthesize carbohydrate differentiation antigen, alloantigens,xenoantigen, or novel determinants detectable by antibody. Examplesinclude the following:

-   -   The marker SSEA-1, for which the effector sequence encodes the        corresponding fucosyltransferase.    -   The Galα(1,3)Gal linkage present on endothelial tissue of most        mammals except for humans and old-world monkeys, formed by an        α(1,3)galactosyltransferase (α1,3GT). The encoding sequence for        sheep and marmoset α1,3GT are provided below (see also Henion et        al., Glycobiology 4:193, 1994). In the processing of human        cells, it is possible to derive a human equivalent α1,3GT, by        correcting the silent human α1,3GT pseudogene (Joziasse et        al., J. Biol. Chem. 266:6991, 1991) using a consensus of the        lower primate α1,3GT sequences.    -   The ABO histo blood group antigens present on most human cells.        The encoding sequence is the corresponding ABO transferases, for        which the encoding sequences are provided below.

Cells displaying the target antigen are separated using a ligand such asan antibody or lectin specific for the target. General techniques usedin producing antibodies and using them in immunoisolation are describedin Handbook of Experimental Immunology (Weir & Blackwell, eds.); CurrentProtocols in Immunology (Coligan et al., eds.); and Methods ofImmunological Analysis (Masseyeff et al., eds., Weinheim: VCH VerlagsGmbH). Polyclonal antibodies can be prepared by injecting a vertebratewith the isolated membrane protein or an immunogenic fragment in asuitable adjuvant. Any unwanted cross-reactivity (such as reactivityagainst proteins expressed by the differentiated cells) can be removedby adsorbing with cross-reacting antigens attached to a solid phase, andcollecting the unbound fraction. Production of monoclonal antibodies isdescribed in such standard references as Harrow & Lane (1988), U.S. Pat.Nos. 4,491,632, 4,472,500 and 4,444,887, and Methods in Enzymology 73B:3(1981). Briefly, a mammal is immunized, their splenocytes areimmortalized, and clones are selected that produce antibody of thedesired specificity. Other methods of obtaining specific antibodymolecules involve contacting a library of immunocompetent cells or viralparticles (such as a phage display library) with the target antigen, andgrowing out positively selected clones (Marks et al., New Eng. J. Med.335:730, 1996; WO 94/13804; WO 92/01047; WO 90/02809; and McGuiness etal., Nature Biotechnol. 14:1449, 1996).

Where the antibody target is a carbohydrate blood group alloantigen orxenoantigen, there are often “naturally occurring” antibodies alreadypresent in the circulation of mammals lacking the antigen without themhaving been deliberately immunized. It is thought that naturallyoccurring antibodies arise from cross-reactive carbohydrates present inthe diet. Humans have naturally occurring antibodies to the human ABOblood group, and to the Galα(1,3)Gal xenoantigen. The “IB4” lectin fromBandeiraea (Griffonia) simplicifolia (Sigma Cat. L 3019) is specific forα-D-galactosyl residues and binds both the Galα(1,3)Gal epitope, and Bblood group substance. Lectins of appropriate specificity can substitutefor antibodies in some affinity separation procedures.

Where the antibody target is a carbohydrate antigen shared betweenspecies, it may be difficult to obtain a high affinity antibody fromanother mammal—in which case, it may be preferable to obtain antibodyfrom an avian species or a phage display library. This invention alsocontemplates artificial carbohydrate antigens, built by using aneffector glycosyltransferase that is not of mammalian origin, butcapable of modifying mammalian complex carbohydrates. Novel carbohydrateantigens may be particularly immunogenic for raising antibody useful indepleting the undifferentiated cells.

In order to effect separation, the cell population is cultured underconditions that allow the transcriptional control element to driveexpression of the effector sequence in undifferentiated cells. The cellpopulation is then subjected to immunoseparation by combining with anantibody or lectin, and then recovering cells that are not bound. Theundifferentiated cells will be in the bound fraction, and will bedepleted from the population recovered. One separation technique isimmunoaffinity binding, in which the antibody is attached to a solidsupport, and the cells are passed over the adsorbent. Differentiatedcells will be in the nonadherent fraction. Another separation techniqueis fluorescence-activated cell sorting, in which the cells are contactedwith a fluorescently labeled primary or secondary antibody. Thedifferentiated cells will be in the non-fluorescent cell sort. Anotherseparation technique is complement-mediated lysis. The cells arecombined with a complete (complement-fixing) antibody, andsimultaneously or sequentially with complement obtained from fresh serumor a commercial source. The complement will lyse undifferentiated cells,depleting them from the population.

As an alternative to ligand targets, the effector gene can encode apeptide toxin—such as ricin, abrin, diphtheria, gelonin, Pseudomonasexotoxin A, and so on. Also suitable are genes that induce or mediateapoptosis—such as the ICE-family of cysteine proteases, the Bcl-2 familyof proteins, Bax, bclXs and caspases. Koga et al. (Hu. Gene Ther.11:1397, 2000) propose a telomerase-specific gene therapy using thehTERT gene promoter linked to the apoptosis gene Caspase-8 (FLICE). Guet al. (Cancer Res. 60:5359, 2000) reported a binary adenoviral systemthat induced Bax expression via the hTERT promoter.

Other suitable effectors encode polypeptides having activity that is notitself toxic to a cell, but renders the cell sensitive to an otherwisenontoxic compound—either by metabolically altering the cell, or bychanging a non-toxic prodrug into a lethal drug. Exemplary is thymidinekinase (tk) , such as may be derived from a herpes simplex virus, whichconverts the anti-herpetic agent ganciclovir (GCV) to a toxic productthat interferes with DNA replication. International Patent PublicationsWO 98/14593 and WO 00/46355 (Geron Corporation) describe constructscomprising HSV tk under control of hTERT promoter sequences. When thetransducing agent is a viral vector, the effector can be a viral generequired for replication of the virus. Essential genes for replicationof adenovirus include the E4, E1a, E1b, and E2 regions. SeeInternational Patent Publication WO 00/46355 (Morin et al., GeronCorporation) for a description of lytic vectors that replicate in cellsexpressing TERT.

Another possible effector sequence encodes a nucleic acid for antisense,a ribozyme or RNA interference (RNAi) targeting transcripts essentialfor cell viability. In one illustration, the hTERT promoter drives RNAIthat inactivates hypoxanthine-guanine phosphoribosyl transferase (HGPRT)or tk. Residual undifferentiated cells can be removed from thepopulation by incubation with medium is supplemented with hypoxanthine,aminopterin, and thymidine (HAT medium).

The vector constructs for use in this invention can also contain apositive selection marker, such as an antibiotic resistance gene, thatis also under control of the specific promoter. Exemplary is a vectorhaving the configuration hTERT promoter—effector gene—IRES or 2Asequence—neo. This is designed so that both the effector and drugresistance gene are expressed under control of the promoter that drivespreferential expression in undifferentiated cells.

Selection Techniques to Eliminate Undifferentiated Cells

To deplete differentiated cell populations of undifferentiated cells,the effector gene is selectively expressed in the undifferentiatedcells.

This can be accomplished in several ways. In one embodiment, thepopulation is genetically altered using a vector in which atranscriptional control element of the appropriate specificity isoperatively linked to the effector gene. The genetic alteration may betransient (for example, using an adenovirus vector), meaning that thelevel of expression diminishes as the cells divide. This is suitable forgenerating differentiated cell populations that will be free ofheterologous genes at the time of therapy. The genetic alteration mayalso be permanent (for example, using a retroviral vector), meaning thatthe alteration is inheritable by progeny of the initially altered cell.This is suitable for generating differentiated cell populations thatwill have an ongoing corrective function as they proliferate in vitro orin vivo, to eliminate any undifferentiated or dedifferentiated cellsthat arise in the population.

Any suitable expression vector can be used. Suitable viral vectorsystems for producing stem cells altered according to this invention canbe prepared using commercially available virus components. Viral vectorscomprising effector genes are generally described in the publicationsreferenced in the last section. Alternatively, vector plasmids can beintroduced into cells by electroporation, or using lipid/DNA complexes,such as those described in U.S. Pat. Nos. 5,578,475; 5,627,175;5,705,308; 5,744,335; 5,976,567; 6,020,202; and 6,051,429. Exemplary isthe formulation Lipofectamine 2000™, available from Gibco/LifeTechnologies. Another exemplary reagent is FuGENE™ 6 TransfectionReagent, a blend of lipids in non-liposomal form and other compounds in80% ethanol, obtainable from Roche Diagnostics Corporation.

In another embodiment, the effector gene is placed under control of anendogenous transcriptional control element, such as the hTERT or OCT-4promoter. This can be effected, for example, by homologousrecombination, using a vector comprising the effector encoding sequence,flanked on one side by the transcriptional control element and otherupstream genomic sequence, and flanked on the other side by downstreamgenomic sequence for the targeted gene. U.S. Pat. Nos. 5,464,764 and5,631,153 describe a double-selection strategy, in which two sequenceshomologous to the gene target flank a positive selection marker, and anegative selection marker is attached to the 3′ terminal of the secondflanking region. U.S. Pat. No. 5,789,215 reports the use of homologousrecombination targeting vectors for modifying the cell genome of mouseembryonic stem cells. Other information of interest for homologousrecombination targeting can be found in U.S. Pat. Nos. 5,589,369,5,776,774, and 5,789,215.

If the effector gene directly causes cell lysis or apoptosis, then thepopulation will be depleted of undifferentiated cells upon culturing thecells under conditions where the control element is expected to causetranscription of the gene. However, if the effector gene is not directlylethal, but renders the cell susceptible to the lethal effects of anexternal agent, then depletion will be postponed until the externalagent is provided. For example, where the gene is a prodrug convertingenzyme, then depletion is effected upon placing the cells in anenvironment containing the prodrug. Where the gene creates an antibodytarget, then depletion is effected upon placing the cells in anenvironment containing specific antibody, plus complement. Theenvironment can be a culture vessel, in which case the agent can just beadded to the culture medium at the requisite concentration.Alternatively or in addition, depletion can be performed in vivo, byadministering the cell population to a subject, and simultaneously orsequentially administering the agent, if not already present. Where thegene is a glycosyltransferase that creates a xenoantigen or analloantigen, then placing the cells in a subject will render the cellsliable to lysis in situ by naturally occurring antibody and complement.

Cell populations in which the majority of cells are differentiated canbe genetically modified according to these procedures to depleteundifferentiated cells. Alternatively, a precursor population ofrelatively undifferentiated cells can be genetically modified accordingto these procedures, and then differentiated. In this situation, it ismore typical to use an effector gene that does not kill the cellsimmediately upon expression, but renders the cells susceptible to thelethal effect of some external agent. In one illustration,undifferentiated pPS cells grown in culture are transduced with anadenovirus vector in which the herpes thymidine kinase gene is undercontrol of the hTERT promoter. The cells are optionally selected forpositive transduction, either by incorporating a selectable marker inthe construct, or by measuring expression of the transduced gene, andproliferated in culture. When differentiated cells are desired, thepopulation is taken through a differentiation procedure (for example, tomake hepatocyte or neuron precursors, as described earlier). They arethen cultured under conditions that permit expression of the tk gene inthe presence of ganciclovir.

Cell populations may be obtained using these techniques that are“depleted” of undifferentiated cells, which indicates any significantreduction in the proportion of undifferentiated cells present. After theprocedure is effected, the proportion of undifferentiated cells may bedecreased by 50% or even 90%. Depending on the control element andeffector chosen, it may be possible to achieve differentiated cellpopulations that are “essentially free” of undifferentiated cells. Thismeans that the population as a whole contains less than 1% of cells withthe undifferentiated phenotype. Populations containing less than 0.2%,0.05%, or 0.01% undifferentiated cells are increasingly more preferred.For pPS cells, the presence of undifferentiated cells can be determinedby counting cells expressing SSEA-4 by FACS analysis, or by countingcells expressing TERT or OCT-4 by fluorescence in-situ hybridization.

Use of Differentiated Cells

Cells prepared according to this invention can be used for a variety ofcommercially important research, diagnostic, and therapeutic purposes.

Because the cell populations of this invention are depleted ofundifferentiated cells, they can be used to prepare antibodies and cDNAlibraries that are specific for the differentiated phenotype. Generaltechniques used in raising, purifying and modifying antibodies areoutlined in the references provided above. General techniques involvedin preparation of mRNA and cDNA libraries are described in RNAMethodologies: A Laboratory Guide for Isolation and Characterization (R.E. Farrell, Academic Press, 1998); cDNA Library Protocols (Cowell &Austin, eds., Humana Press); and Functional Genomics (Hunt & Livesey,eds., 2000).

Relatively homogeneous cell populations are particularly suited for usein drug screening and therapeutic applications.

Drug Screening

Differentiated pPS cells of this invention can be used to screen forfactors (such as solvents, small molecule drugs, peptides,polynucleotides, and the like) or environmental conditions (such asculture conditions or manipulation) that affect the characteristics ofdifferentiated cells.

In some applications, differentiated cells are used to screen factorsthat promote maturation, or promote proliferation and maintenance ofsuch cells in long-term culture. For example, candidate maturationfactors or growth factors are tested by adding them to pPS cells indifferent wells, and then determining any phenotypic change thatresults, according to desirable criteria for further culture and use ofthe cells.

Particular screening applications of this invention relate to thetesting of pharmaceutical compounds in drug research. The reader isreferred generally to the standard textbook “In vitro Methods inPharmaceutical Research”, Academic Press, 1997, and U.S. Pat. No.5,030,015). Assessment of the activity of candidate pharmaceuticalcompounds generally involves combining the differentiated cells of thisinvention with the candidate compound, determining any change in themorphology, marker phenotype, or metabolic activity of the cells that isattributable to the compound (compared with untreated cells or cellstreated with an inert compound), and then correlating the effect of thecompound with the observed change.

The screening may be done, for example, either because the compound isdesigned to have a pharmacological effect on certain cell types, orbecause a compound designed to have effects elsewhere may haveunintended side effects. Two or more drugs can be tested in combination(by combining with the cells either simultaneously or sequentially), todetect possible drug-drug interaction effects. In some applications,compounds are screened initially for potential toxicity (Castell et al.,pp. 375-410 in “In vitro Methods in Pharmaceutical Research,” AcademicPress, 1997). Cytotoxicity can be determined in the first instance bythe effect on cell viability, survival, morphology, and expression orrelease of certain markers, receptors or enzymes. Effects of a drug onchromosomal DNA can be determined by measuring DNA synthesis or repair.[³H]-thymidine or BrdU incorporation, especially at unscheduled times inthe cell cycle, or above the level required for cell replication, isconsistent with a drug effect. Unwanted effects can also include unusualrates of sister chromatid exchange, determined by metaphase spread. Thereader is referred to A. Vickers (PP 375-410 in “In vitro Methods inPharmaceutical Research,” Academic Press, 1997) for further elaboration.

Therapeutic Use

Differentiated cells of this invention can also be used for tissuereconstitution or regeneration in a human patient in need thereof. Thecells are administered in a manner that permits them to graft to theintended tissue site and reconstitute or regenerate the functionallydeficient area.

In one example, neural stem cells are transplanted directly intoparenchymal or intrathecal sites of the central nervous system,according to the disease being treated. Grafts are done using singlecell suspension or small aggregates at a density of 25,000-500,000 cellsper μL (U.S. Pat. No. 5,968,829). The efficacy of neural celltransplants can be assessed in a rat model for acutely injured spinalcord as described by McDonald et al. (Nat. Med. 5:1410, 1999. Asuccessful transplant will show transplant-derived cells present in thelesion 2-5 weeks later, differentiated into astrocytes,oligodendrocytes, and/or neurons, and migrating along the cord from thelesioned end, and an improvement in gate, coordination, andweight-bearing.

Certain neural progenitor cells embodied in this invention are designedfor treatment of acute or chronic damage to the nervous system. Forexample, excitotoxicity has been implicated in a variety of conditionsincluding epilepsy, stroke, ischemia, Huntington's disease, Parkinson'sdisease and Alzheimer's disease. Certain differentiated cells of thisinvention may also be appropriate for treating dysmyelinating disorders,such as Pelizaeus-Merzbacher disease, multiple sclerosis,leukodystrophies, neuritis and neuropathies. Appropriate for thesepurposes are cell cultures enriched in oligodendrocytes oroligodendrocyte precursors to promote remyelination.

Hepatocytes and hepatocyte precursors prepared according to thisinvention can be assessed in animal models for ability to repair liverdamage. One such example is damage caused by intraperitoneal injectionof D-galactosamine (Dabeva et al., Am. J. Pathol. 143:1606, 1993).Efficacy of treatment can be determined by immunohistochemical stainingfor liver cell markers, microscopic determination of whether canalicularstructures form in growing tissue, and the ability of the treatment torestore synthesis of liver-specific proteins. Liver cells can be used intherapy by direct administration, or as part of a bioassist device thatprovides temporary liver function while the subject's liver tissueregenerates itself following fulminant hepatic failure.

The efficacy of cardiomyocytes prepared according to this invention canbe assessed in animal models for cardiac cryoinjury, which causes 55% ofthe left ventricular wall tissue to become scar tissue without treatment(Li et al., Ann. Thorac. Surg. 62:654, 1996; Sakai et al., Ann. Thorac.Surg. 8:2074, 1999, Sakai et al., J. Thorac. Cardiovasc. Surg. 118:715,1999). Successful treatment will reduce the area of the scar, limit scarexpansion, and improve heart function as determined by systolic,diastolic, and developed pressure. Cardiac injury can also be modeledusing an embolization coil in the distal portion of the left anteriordescending artery (Watanabe et al., Cell Transplant. 7:239, 1998), andefficacy of treatment can be evaluated by histology and cardiacfunction. Cardiomyocyte preparations embodied in this invention can beused in therapy to regenerate cardiac muscle and treat insufficientcardiac function (U.S. Pat. No. 5,919,449 and WO 99/03973).

The examples that follow are provided by way of further illustration,and are not meant to imply any limitation in practicing the claimedinvention.

EXAMPLES Example 1 Feeder-Free Passage of hES Cells

In this experiment, undifferentiated hES cells that had been maintainedon primary mouse embryonic feeder cells were harvested, and thenmaintained in the absence of feeders. The culture wells were coated withMatrigel®, and the cells were cultured in the presence of conditionednutrient medium obtained from a culture of irradiated primaryfibroblasts.

Preparation of Conditioned Media (CM) from Primary Mouse EmbryonicFibroblasts (mEF):

Fibroblasts were harvested from T150 flasks by washing one time withCa⁺⁺/Mg⁺⁺ free PBS and incubating in 1.5-2 mL trypsin/EDTA (Gibco) forabout 5 min. After the fibroblasts detached from the flask, they werecollected in mEF media (DMEM+10% FBS). The cells were irradiated at 4000rad (508 sec at 140 kV: shelf setting 6 in a Torrex generator), countedand seeded at about 55,000 cells cm⁻² in mEF media (525,000 cells/wellof a 6 well plate). After at least 4 hours the media were exchanged withSR containing ES media (containing bFGF), using 3-4 mL per 9.6 cm wellof a 6 well plate. Conditioned media was collected daily for feeding ofhES cultures. Alternatively, medium was prepared using mEF plated inculture flasks, exchanging medium daily at 0.3-0.4 mL cm⁻². Beforeaddition to the hES cultures, the conditioned medium was supplementedwith 4 ng/mL of human bFGF (Gibco). Fibroblast cultures were used inthis system for about 1 week, before replacing with newly preparedcells.

Matrige® Coating:

Growth Factor Reduced Matrigel® or regular Matrigel® (Becton-Dickinson,Bedford Mass.) was thawed at 4° C. The Matrigel® was diluted 1:10 to1:500 (typically 1:30) in cold KO DMEM. 0.75-1.0 mL of solution wasadded to each 9.6 cm² well, and incubated at room temp for 1 h. Thecoated wells were washed once with cold KO DMEM before adding cells.Plates were used within 2 h after coating, or stored in DMEM at 4° C.and used within ˜1 week.

Human ES Culture:

Undifferentiated hES colonies were harvested from hES cultures onfeeders as follows. Cultures were incubated in ˜200 U/mL collagenase IVfor about 5 minutes at 37° C. Colonies were harvested by pickingindividual colonies up with a 20 μL pipet tip under a microscope or byscraping and dissociating into small clusters in conditioned medium(CM). These cells were then seeded onto Matrigel® in conditioned mediaat 15 colonies to each 9.6 cm² well (if 1 colony is ˜10,000 cells, thenthe plating density is ˜15,000 cells cm⁻²).

The day after seeding on Matrigel®, hES cells were visible as smallcolonies (˜100-2,000 cells) and there were single cells in-between thecolonies that appeared to be differentiating or dying. As the hES cellsproliferated, the colonies became quite large and very compact,representing the majority of surface area of the culture dish. The hEScells in the colonies had a high nucleus to cytoplasm ratio and hadprominent nucleoli, similar to hES cells maintained on feeder cells. Atconfluence, the differentiated cells in between the colonies representedless than 10% of the cells in the culture.

Six days after seeding, the cultures had become almost confluent. Thecultures were split by incubating with 1 mL ˜200 U/mL Collagenase IVsolution in KO DMEM for ˜5 minutes at 37° C. The collagenase solutionwas aspirated, 2 mL hES medium was added per well, and the hES cellswere scraped from the dish with a pipette. The cell suspension wastransferred to a 15 mL conical tube, brought up to a volume of 6 mL, andgently triturated to dissociate the cells into small clusters of 10-2000cells. The cells were then re-seeded on Matrigel® coated plates in CM,as above. Cells were seeded at a 1:3 or 1:6 ratio, approximately 90,000to 170,000 cells cm⁻², making up the volume in each well to 3 mL. Mediumwas changed daily, and the cells were split and passaged again at 13 dand again at 19 d after initial seeding.

On day 19 after initial seeding, cells were harvested and evaluated forsurface marker expression by immunofluorescence cell cytometry, usinglabeled antibodies specific for cell surface markers. For the hES cellsmaintained in the absence of feeders, a high percentage express SSEA-4,Tra-1-60 or Tra-1-81. These 3 marke are expressed on undifferentiatedhuman ES cells that are maintained on feeders (Thomson et al., 1998). Inaddition, there is very little expression of SSEA-1, a glycolipid thatis not expressed(or expressed at low levels) on undifferentiated EScells. Immunohistochemical evaluation of SSEA-4, Tra-1-60 and Tra-1-81indicates that the expression of these markers is localized to the EScolonies, not the differentiated cells in between the colonies.

Cultures of hES cells have been grown in the absence of feeder cells forover 180 days after initial seeding, with no apparent change in theproliferative capacity or phenotype. Human ES cells maintained onMatrigel® in mEF conditioned medium have a doubling time of about 31-33hours, similar to the proliferation rate for hES cells grown on mEFfeeder cells. H1 cells after 64 days of feeder-free culture showed anormal karyotype.

Example 2 Phenotypic Markers of hES Cells in Feeder-free Culture

Undifferentiated hES cells express SSEA-4, Tra-1-60, Tra-1-81, OCT-4,and hTERT. The expression of these markers decreases upondifferentiation. In order to assess whether the cells maintained infeeder-free conditions retained these markers, cells were evaluated byimmunostaining, reverse transcriptase PCR amplification, and assay fortelomerase activity.

For analysis by fluorescence-activated cell sorting (FACS), the hEScells were dissociated in 0.5 mM EDTA in PBS and resuspended to about5×10⁵ cells in 50 μL diluent containing 0.1% BSA in PBS. For analyzingsurface marker expression, cells were incubated in the primaryantibodies, including IgG isotype control (0.5 μg/test), IgM isotypecontrol (1:10), SSEA-1 (1:10), SSEA-4 (1:20), Tra-1-60 (1:40) andTra-1-81 (1:80), diluted in the diluent at 4° C. for 30 min. Afterwashing with the diluent, cells were incubated with rat anti-mouse kappachain antibodies conjugated with PE (Becton Dickinson, San Jose, Calif.)at 4° C. for 30 min. Cells were washed and analyzed on FACScalibur™ FlowCytometer (Becton Dickinson, San Jose, Calif.) using CellQuest™software.

Similar to the hES cells on feeders, cells on Matrigel®, laminin,fibronectin or collagen IV expressed SSEA-4, Tra-1-60 and Tra-1-81.There was very little expression of SSEA-1, a glycolipid that is notexpress undifferentiated hES cells.

For analysis by immunocytochemistry, cells were incubated with primaryantibodies, including SSEA-1 (1:10), SSEA-4 (1:20), Tra-1-60 (1:40) andTra-1-81 (1:80), diluted in knockout DMEM at 37° C. for 30 min. Cellswere then washed with warm knockout DMEM and fixed in 2%paraformaldehyde for 15 min. After washing with PBS, cells wereincubated with 5% goat serum in PBS at room temp for 30 min, followed byincubation with the FITC-conjugated goat anti-mouse antibodies (1:125)(Sigma) at room temp for 30 min. Cells were washed, stained with DAPIand mounted. The staining was typically performed ˜2 days afterpassaging. Cells were also examined for expression of alkalinephosphatase, a marker for undifferentiated ES cells. This was performedby culturing the cells on chamber slides, fixing with 4%paraformaldehyde for 15 min, and then washing with PBS. Cells were thenincubated with alkaline phosphatase substrate (Vector Laboratories,Inc., Burlingame, Calif.) at room temperature in the dark for 1 h.Slides were rinsed for 2-5 min in 100% ethanol before mounting.

The results showed that SSEA-4, Tra-1-60, Tra-1-81, and alkalinephosphatase were expressed by the hES colonies on Matrigel® or laminin,as seen for the cells on feeders—but not by the differentiated cells inbetween the colonies.

FIG. 1 shows OCT-4 and hTERT expression of H1 cells on feeders and offfeeders, as detected by reverse-transcriptase PCR amplification. Forradioactive relative quantification of individual gene products,QuantumRNA™ Alternate18S Internal Standard primers (Ambion, Austin Tex.,USA) were employed according to the manufacturer's instructions.Briefly, the linear range of amplification of a particular primer pairwas determined, then coamplified with the appropriate mixture ofalternate18S primers:competimers to yield PCR products with coincidinglinear ranges. Before addition of AmpliTaq™ (Roche) to PCR reactions,the enzyme was pre-incubated with the TaqStar™ antibody (ProMega)according to manufacturer's instructions. Radioactive PCR reactions wereanalyzed on 5% non-denaturing polyacrylamide gels, dried, and exposed tophosphoimage screens (Molecular Dynamics) for 1 hour. Screens werescanned with a Molecular Dynamics Storm 860 and band intensities werequantified using ImageQuant™ software. Results are expressed as theratio of radioactivity incorporated into the hTERT or OCT-4 band,standardized to the radioactivity incorporated into the 18s band.

Primers and amplification conditions for particular markers are asfollows. OCT-4: Sense (SEQ. ID NO:2) 5′-CTTGCTGCAG AAGTGGGTGGAGGAA-3′AntiSense (SEQ. ID NO:3) 5′-CTGCAGTGTG GGTTTCGGGC A-3′;alternate18:competimers 1:4; 19 cycles (94° 30 sec; 60° 30 sec; 72° 30sec). hTERT: Sense (SEQ. ID NO:4) 5′-CGGAAGAGTG TCTGGAGCAA-3′AntiSense(SEQ. ID NO:5) 5′-GGATGAAGCG GAGTCTGGA-3′; alternate18:competimers 1:12;34 cycles (94° 30 sec; 60° 30 sec; 72° 30 sec).

hTERT and OCT-4 expression was seen in all the culture conditions exceptMatrigel® and regular medium. Furthermore, after exposure of cells toretinoic acid (RA) or dimethyl sulfoxide (DMSO), factors that promotecell differentiation, the expression of hTERT was markedly decreased.

FIG. 2 shows telomerase activity measured by TRAP activity assay (Kim etal., Science 266:2011, 1997; Weinrich et al., Nature Genetics 17:498,1997). All the cultures conditions showed positive telomerase activityafter 40 days on Matrigel®, laminin, fibronectin or collagen IV in mEFconditioned medium.

Example 3 Differentiation of hES Cells

In this experiment, differentiation using standard methods of aggregateformation was compared with a direct differentiation technique.

For the aggregate differentiation technique, monolayer cultures ofrhesus and human ES lines were harvested by incubating in Collagenase IVfor 5-20 min, and the cells were scraped from the plate. The cells werethen dissociated and plated in non-adherent cell culture plates inFBS-containing medium. The plates were placed into a 37° C. incubator,and in some instances, a rocker was used to facilitate maintainingaggregates in suspension. After 4-8 days in suspension, aggregate bodiesformed and were plated onto a substrate to allow for furtherdifferentiation.

For the direct differentiation technique, suspensions of rhesus andhuman ES cells were prepared in a similar fashion. The cells were thendissociated by trituration to clusters of ˜50-100 cells, and plated ontoglass coverslips treated with poly-ornithine. The cells were maintainedin serum containing medium, or defined medium for 7-10 days beforeanalysis.

Cells from both preparations were fixed and tested by immunoreactivityfor β-tubulin III and MAP-2, which is characteristic of neurons, andglial fibrillary acidic protein (GFAP), which is characteristic ofastrocytes. Results are shown in Table 1.

TABLE 1 Comparison of hPS Differentiation Methods Differentiation viaDirect ES Cell Line Aggregate Bodies Differentiation used fordifferentiation Neurons Astrocytes Neurons Astrocytes R366.4 (Rhesusline) + + + + R278.5 (Rhesus line) + + + + R456 (Rhesus line) + + + + H9(Human line) + + + + H9.1 (Clone of (Not Done) (Not Done) + + H9) H9.2(Clone of + + + + H9)

Rhesus and human ES lines differentiated into cells bearing markers forneurons and astrocytes, using either the aggregate or directdifferentiation technique. In the rhesus cultures, percentage ofaggregates that contained neurons ranged from 49% to 93%. In the humanlines examined, the percentage of aggregates containing neurons rangedfrom 60% to 80%. Double labeling for GABA and β-tubulin indicated that asub-population of the neurons express the inhibitory neurotransmitterGABA. In addition, astrocytes and oligodendrocytes were identified withGFAP immune reactivity and GalC immune reactivity, respectively.Therefore, the human and rhesus ES cells have the capacity to form allthree major cell phenotypes in the central nervous system.

The effect of several members of the neurotrophin growth factor familywas examined. hES cells were differentiated by harvesting withcollagenase, dissociating, and reseeding onto poly-ornithine coatedcover slips. The cells were plated into DMEM/F12+N2+10% FBS overnight.The following day, the serum was removed from the medium and replacedwith 10 ng/mL human bFGF and the growth factor being tested. After 24hours, bFGF was removed from the medium. These cultures were fed everyother day. They were fixed after 7 days of differentiation andimmunostained for analysis. The number of neurons was evaluated bycounting cells positive for β-tubulin. Cultures maintained in thepresence of 10 ng/mL brain derived neurotrophic factor (BDNF) formedapproximately 3-fold more neurons than the control cultures. Culturesmaintained in neurotrophin-3 (1 ng/mL) formed approximately 2-fold moreneurons than control cultures.

In a subsequent experiment, suspensions of human ES cells were preparedfrom parental line H9 and two subcloned lines. The cells were harvestedusing collagenase IV, and then replated onto poly-ornithine coated glassslides in medium containing 20% FBS. The cultures were then fed everyother day for 7-10 days, then fixed for immunostaining. From each ofthese lines, a number of differentiated cells stained positively formuscle-specific actin (antibody from Dako), but were negative forcardiac troponin I. Several patches of cells stained positively forα-fetoprotein, indicating the presence of endoderm cells.

Example 4 Comparison of Direct Differentiation with DifferentiationThrough Embryoid Bodies

To induce direct differentiation, undifferentiated hES cells wereharvested and re-plated directly into differentiating conditions.Considerable cell death was apparent upon plating, but many cellsadhered and began to proliferate and/or differentiate. In culturesdifferentiated using serum containing conditions, the cultures continuedto proliferate and reached confluence within 5-10 days. At this time,the cultures contained a heterogeneous population that displayed manydifferent morphologies. Immunocytochemistry revealed ectoderm, mesodermand endoderm lineages using antibodies against β-tubulin III, musclespecific actin and α-fetoprotein, respectively. The positive stainingfor all of these cell types appeared in patches that were sometimesquite dense, therefore it was difficult to accurately quantify thepercentages of each cell type.

In order to increase the percentage of neurons, the hES cells wereplated onto poly-ornithine coated glass coverslips and cultures indefined media. Although these data indicate that cells from all threegerm layers can be derived without the production of EBs, cardiomyocyteswere not identified.

By way of comparison, hES cells were induced to differentiate bygenerating embryoid bodies (EBs). In these experiments, ES cells wereharvested and replated in suspension cultures. Although initially amarked amount of cell death was observed, after 2-3 days the remainingcells formed aggregates. EBs were maintained for as many as 16 days inculture and were still viable and formed many structures aftersubsequent plating. Later stage human EBs often showed a cysticmorphology and sometimes gave rise to beating EBs.

To assess cardiomyocyte formation, EBs were transferred togelatin-coated plates or chamber slides after 4 days in the suspensioncultures. The EBs attached to the surface after seeding, proliferatedand differentiated into different types of cells. Spontaneouslycontracting cells were observed in various regions of the culture atdifferentiation day 8 and the number of beating regions increased untilabout day 10. In some cases, more than 75% of the EBs had contractingregions. Beating cells were morphologically similar to mouse EScell-derived beating cardiomyocytes. In addition, the expression of thecardiac specific marker cardiac troponin 1 was examined atdifferentiation day 15 using immunocytochemistry. Individual contractingfoci in the differentiated cultures were photographed to record thecontracting area before the culture was fixed. The culture was thenevaluated for cardiac cTnl expression and matched to the originalphotographs to determine the percentage of contracting areas that werepositive for cTnl staining. As a control, cells adjacent to thecontracting foci were also examined for cTnl staining. In these cultures100% of the contracting areas showed positive immunoreactivity, whileminimal immunoreactivity was observed in the non-beating cells.

Cultures of differentiated EBs were subjected to Western blot analysisusing monoclonal antibody against cTnl. This assay gave a strong 31 kDaprotein signal, corresponding to the size of the purified native humancardiac Tnl. cTnl was detected in differentiated human ES cellscontaining contracting cells but not in undifferentiated ES cells ordifferentiated cultures with no evidence of contracting cells,suggesting the specific detection of cardiomyocytes. As a control, theblot was reprobed with β-actin specific antibody, confirming thepresence of similar amounts of proteins in all samples.

In other experiments, EBs were cultured for 8 or 16 days and maintainedas adherent cultures for an additional 10 days. RNA was prepared fromthe differentiated human ES cells and semiquantitative RT-PCR wasperformed to detect the relative expression of the endoderm-specificproducts α₁-anti-trypsin, AFP, and albumin. Low levels ofα₁-anti-trypsin and AFP were detected in the undifferentiated cultures;little or no albumin was detected in the same cultures. All 3 markerswere detected at significantly higher levels after differentiation.Expression of all 3 endoderm markers was higher in cultures derived from8 day embryoid bodies than 16 day embryoid bodies.

Example 5 Transfection and Transduction of hES Cells Maintained onPrimary mEF Feeder Layers

hES cultures were maintained in a growth medium composed of 80% KO DMEM(Gibco) and 20% Serum Replacement (Gibco) supplemented with 1%non-essential amino acids, 1 mM glutamine, 0.1 mM β-mercaptoethanol and4 ng/mL hbFGF (Gibco).

Plates were coated with a solution of 0.5% gelatin (Sigma) at 37°overnight prior to the addition of cells. Primary mEFs were cultured instandard mEF medium, and split 1:2 every 2 days for up to 5 splits.Subconfluent cultures of mEFs were detached with trypsin, resuspended in10 mL medium, and irradiated with a cumulative dose of 3500-4000 radswith a Torrex 150D X-ray generator. Irradiated cells were pelleted at400×g for 5 min and resuspended at 1.25×10⁵ cells per mL in standard mEFmedium. Individual wells of a 6-well plate were seeded with 3.75×10⁵irradiated mEFs per well; individual wells of a 24-well plate wereseeded with 75,000 irradiated mEFs per well.

Transfection was performed as follows. hES cells plated in 6 well plateswere removed from the feeder layer with collagenase (˜200 units/mL) at37° for 7-10 min. When colonies began to detach, the collagenase fromeach well was aspirated and replaced with 2 mL of standard hES growthmedium/well. The hES cells were removed by scraping the surface of asingle well with a 5 mL pipet and transferred to a 50 mL conical tube.Additional hES growth medium was added to a final volume of 10 mL. Thecell suspension was triturated 10-12 times with a 10 mL pipet, and anadditional 8 mL of standard hES growth medium added. Three mL of thecell suspension were added to each well of 6 well plates that werepre-coated with gelatin and mEF feeder layers as described above (i.e.,1 well of a 6 well plate was sufficient to seed 6 wells of a new plate).

Replated hES cells were tested with a number of different transfectionsystems to determine whether genetic alteration of hES cells could beachieved without causing differentiation. Systems tested included thefollowing: Mammalian Transfection Kit (CaPO4 and DEAE reagents),Stratagene cat # 200285; TranslT-LT1 Mirus (Panvera), cat # MIR 2310;Polybrene (Sigma); Poly-L-Lysine (Sigma); Superfect™ (Qiagen);Effectene™ (Qiagen); Lipofectin™ (Life Technologies); Lipofectamine(differs from Lipofectamine 2000™) (Life Technologies); Cellfectin™(Life Technologies); DMRIE-C (Life Technologies); Lipofectamine 2000(Life Technologies); and electroporation using BioRad™ Gene pulser.

Under the conditions used, Lipofectamine 2000™ (Gibco Life Technologiescat # 11668019, patent pending) and FuGENE™ (trademark of Fugent L.L.C.;a proprietary blend of lipids and other components, purchased from RocheDiagnostic Corporation cat # 1 814 443) both resulted in goodtransfection efficiency. The efficiency was generally best if thesereagents were contacted with replated hES cells ˜48 h after thereplating.

Transfection using Lipofectamine 2000™ was conducted as follows: Theplasmid DNA (3-5 μg of pEGFP-C1, ClonTech cat. # 6084-1) was diluted inwater to a final volume of 100 μl. In pilot experiments, 5 to 30 μL ofLipofectamine 2000™ (Gibco, cat # 11668-019) were diluted in OptiMEM™(Gibco, cat # 11-58-021) to a final volume of 100 μL. The DNA solutionwas then added slowly to the Lipofectamine2000™ solution and mixedgently. The mixture was incubated at room temperature for 20-30 minbefore being supplemented with 800 μl of OptiMEM™ . Cells were washedwith 3 mL of pre-warmed OptiMEM™ and incubated in 0.5-1 mL of theDNA/lipid mixture solution at 37° C. for 4 h, per well (9.6 cm²). Insome experiments, after 4 h, the complex was removed before the additionof 4 mL of mEF-conditioned medium; in others, sufficient mEF-conditionedmedium was added to the wells to reach a final volume of 3.5 mL and themixture was left on the cells overnight. In other experiments theDNA/lipid mixture was added to wells containing sufficientmEF-conditioned medium such that the final volume was 3.5 mL, and thecells were incubated in this mixture overnight.

Transfection using FUGENE™ was conducted as follows. Each well wastransfected with 10 μg DNA using FuGENE™ 6 (Roche Diagnostics Corp.), ata ratio of 3:2 FuGENE™ reagent to DNA as described by the manufacturer'sdirections. OptiMEM™ serum-free medium was used in the transfections. Inthe “old protocol”, 4 h after the addition of the FuGENE™-DNA complex,2.5 mL of standard hES growth medium was added to each transfected well.In the revised protocol (“3:2 L”), transfected wells were not re-fedwith standard hES growth medium. Twenty-four hours after transfection,GFP-expression was assessed by flow cytometry.

Forty-eight hours before transfection, hES cells were seeded onto 6 wellplates that had been coated with gelatin and mEF feeder layers asdescribed above. hES cells were transfected using FuGENE™ 6 (Roche) orLipofectamine 2000™ (Gibco) according to the manufacturers'instructions. Twenty-four hours after transfection, cells were assessedfor GFP expression by inspection under a fluorescent microscope or flowcytometry. In the experiment shown in FIG. 1, three methods werecompared: the standard Lipofectamine 2000™ protocol, the standardFuGENE™ protocol, and a variant FuGENE™ protocol in which the DNA/lipidmix was left on the cells overnight. The results demonstrated that whileLipofectamine 2000™ consistently yielded a higher percentage ofGFP-expressing cells, the variant FuGENE™ protocol resulted inGFP-expressing cells with a higher mean fluorescence intensity.

Transient transductions using adenoviral vectors were conducted asfollows. The vector Ad5CMV5-GFP (referred to here as Ad5GFP) containsthe green fluorescent protein encoding region under control of the CMVpromoter, and was purchased from Quantum Biotechnologies, cat # ADV0030.Seventy-two hours before transduction, hES cells were seeded onto 24well plates that had been coated with gelatin and mEF feeder layers asdescribed above. Before transduction, 3 wells of hES cells were detachedwith a solution of 0.05% trypsin/5 mM EDTA (Sigma) at 37°, resuspendedin 500 μL of standard mEF growth medium, and counted with ahemocytometer (the 75,000 mEF feeder cells were subtracted from eachwell) to establish the cell number before transfection. The adenovirusstock was thawed on ice immediately prior to use.

For infection with Ad5GFP, growth media was aspirated from the wellscontaining hES cells and replaced with 1 mL of hES growth medium plus 9μL of AdS GFP stock (MOI of 40). Two hours later, the virus-containingmedium was replaced with 1 mL of hES growth medium per well. Eachtransduced well was refed with 1 mL of fresh hES growth medium every 24hours. GFP expression was assessed by flow cytometry. The results from atypical experiment indicated that expression was highest at 24 hr aftertransduction but persisted for at least 8 days at low levels (by thelater time points, extensive differentiation had occurred due toovergrowth of the hES cells).

Example 6 Preparation of the Immortalized Feeder Cell Line NH190

In this example, a permanent mouse cell line was established that issuitable for conditioning medium for the culture of primate pluripotentstem (pPS) cells. The NHG190 line is a mouse embryonic fibroblast cellline immortalized with telomerase that is triple drug resistant, andexpresses green fluorescent protein (GFP).

Two mouse strains were obtained from Jackson Laboratory (Bar Harbor,Me.) that have a transgene for resistance to the antibiotics neomycin orhygromycin. The C57BL/6J TgN(pPGKneobpA)3Ems mice andC57BL/6J-TgN(pPWL512hyg)1 Ems mice from Jackson Labs were cross-bred.Embryos that were both neomycin- and hygromycin-resistant were dissectedat day 13.5 post conception according to standard protocols forpreparing mouse embryonic fibroblasts (mEF) for feeder layers (E. J.Robertson, pp. 71-112 in Teratocarcinoma and Embryonic Stem Cell Lines,ed. E. J. Robertson, Oxford: IRL Press, 1987). The derived mEF cellswere stored frozen.

The mEFs were thawed in growth medium containing 20% fetal calf serum(HyClone), 2 mM L-glutamine (Gibco/BRL), 80% DMEM (Gibco/BRL). The cellswere expanded using 1:2 split ratios for 4 passages. Two flasks that hadreached ˜75% confluence were fed with fresh medium 4 h beforeelectroporation. Cells were removed from the flasks with 0.5%trypsin/500 mM EDTA (Gibco/BRL), pelleted at 400×g for 5 min at roomtemperature, and resuspended in the growth medium at a concentration of4×10⁶ cells/mL.

The cell suspension was divided into two 500 μL aliquots and transferredto two 0.4 cm gap electroporation cuvettes (BioRad). One cuvettereceived 5 μg of the control plasmid (pBS212; puromycin-resistance genedriven by the SV40 early enhancer/promoter); the other received 5 μg ofpGRN190, comprising the murine telomerase reverse transcriptase (mTERT)coding region driven by MPSV promoter plus puromycin resistance genedriven by the SV40 early enhancer/promoter. The cells and DNA were mixedby hand, and electroporated using a BioRad gene Pulser with a BioRadcapacitance extender at a setting of 300V, 960 μF.

Each aliquot of cells was transferred to an individual 150 cm platecontaining 25 mL of growth medium. The medium on the plates wasexchanged on the following day, and on the next day, growth medium wasreplaced by growth medium plus 0.5 μg/mL puromycin. The medium on theplates was exchanged for fresh puromycin-containing medium every 48 hrsuntil 29 days after electroporation. At this time, large individualcolonies of puromycin-resistant cells were evident in both the pBS212-and pGRN190- electroporated plates. Ten colonies from the control plateand 12 from the pGRN190-electroporated plate were isolated with cloningcylinders and each colony was transferred to 1 well of a 48-well plate(1 well per colony).

One week later, all surviving colonies that had expanded to reachconfluence in the 48 well plate (three control colonies, 1pGRN190-electroporated colony) were transferred individually to wells ofa 24 well plate. Six days later, the only colony that had continued toexpand was derived from the pGRN190-electroporated plate, and wassubsequently designated NH190. The cells were maintained in growthmedium plus 0.5 μg/mL puromycin. Analysis for telomerase activity byTRAP assay (Kim et al., Nucleic Acids Res. 25:2595, 1997) demonstratedthat NH190 cells express functional telomerase activity.

To facilitate monitoring of the cells in mixed culture populations andin vivo, NH190 cells were further infected with a retroviral constructconferring expression of green fluorescent protein (GFP). The enhancedGFP sequence from plasmid pEGFP-1 is one of the Living Colors™fluorescent protein vectors, available from ClonTech. It contains anenhanced GFP encoding region, with changes that alter restrictionnuclease cleavage sites, and shift the excitation and emissionwavelengths of the encoded protein. The EGFP-1 sequence was cloned intothe vector pMSCV.neo, ClonTech cat # K1062-1. NH190 cells weretransduced with the engineered vector, and GFP positive cells wereseparated by FACS sorting. The GFP expressing cell line was designatedNHG190. These cells have been carried in culture for over 3 months.

Example 7 Genetic Modification of hES Cells Maintained on NHG190 FeederCells

NHG190 cells were cultured in DMEM (Gibco) plus 20% fetal bovine serum(HyClone) and 5 mM glutamine. Cells were split 1:10 every 3 d.Subconfluent cultures were detached with trypsin, suspended in 10 mLmedium, and irradiated with a cumulative dose of 3500 rads with a Torrex150D X-ray generator. Irradiated cells were pelleted at 400×g for 5 minand resuspended at 1.25×10⁵ cells per mL in either NHG190 medium orstandard hES medium.

Conditioned medium was prepared by plating NHG190 cells at 4.08×10⁴ cm⁻¹on gelatin-coated plates. At 18-24 h after plating, medium was exchangedfor standard hES medium with 4 ng/mL added bFGF. The medium wasconditioned by the cells for 18-24 h, harvested, and an additional 4ng/mL bFGF was added. The medium was used to support hES cell culturesthe same day as it was collected. Irradiated NHG190 cells could be usedfor preparing conditioned medium for 7-10 days.

hES cells were transfected as follows. The cells were removed from thefeeder layer using collagenase (˜200 U/mL) at 37° C. for 7-10 min, andtransferred to a 50 mL conical tube. hES growth medium was added to afinal volume of 10 mL; the suspension was triturated 10-12 times with a10 mL pipet, and another 8 mL hES medium was added. Three mL of cellsuspension was added to each well in a 6-well plate precoated withMatrigelQR and NHG190 feeder cells.

Forty-eight hours after seeding, the hES were transfected with 10 μg DNAper well using FuGENE™ 6 (Roche) according to manufacturer's protocol inOptiMEM™ serum-free medium. The DNA was a plasmid containing the PGKpromoter driving neo^(r). Four h later, 3 mL of NHG190-conditionedmedium was added to each transfected well. Cells were re-fed daily with3 mL conditioned medium. Forty-eight h after transfection, the cellswere layered with NHG190 conditioned medium containing 200 μg/mL addedgeneticin (Sigma), which was replaced daily thereafter. After 3 days ofselection, additional irradiated NHG190 feeder cells were added(1.25×10⁵ cells/well in hES medium). Twenty-four h later, the medium wasagain replaced with NHG190-conditioned medium containing 200 μg/mLgeneticin, replaced daily.

Individual colonies were isolated and expanded through another round ofselection. After a further 5 days, individual colonies were identifiedby microscope and marked on the outside of the dish. Medium was removed,and replaced with collagenase (˜200 U/mL). Individual colonies werepicked using a p20 pipet tip, and transferred to individual tubescontaining 2 mL NHG190 conditioned medium (without geneticin). Thesuspension was triturated 5 times to disaggregate colonies, and thecontents of each tube were transferred to a well of a 12-well platecoated with gelatin and irradiated NHG190 cells (1.875×10⁵ cells/well).Cells were fed 24 h later with 2 mL fresh conditioned medium. Two daysafter seeding, cells were layered with 2 mL conditioned mediumcontaining 200 μg/mL geneticin, replaced daily for 5 days. As each wellbecame 50-75% confluent, the cells were detached with collagenase,transferred to 6 mL conditioned medium, and triturated 10-12 times. 3 mLcell suspension was added to each of 2 wells of a 6-well plated coatedwith gelatin and irradiated NHG190 cells (3.75×10⁵ cells/well); thecells were refed with 3 mL conditioned medium at 24 h. The cells werethen selected for 5 days using 3 mL conditioned medium containinggeneticin, and split 1:6 as before.

Stable transduction using retrovirus was conducted as follows.Retroviral vector designated GRN354 was constructed at Geron Corp. usingPMSCVneo vector purchased from ClonTech (cat # K1062-1). The eGFPencoding region was inserted downstream from the MSCV LTR. The LTRdrives expression of GFP and the vector also contains the neo^(r) genedriven by the murine PGK promoter. Plates were coated with 0.5% gelatinand NHG190 feeder cells (7.5×10⁴ in 1 mL NHG190 medium for 24 wellplates; 3.75×10⁵ in 3 plates). The hES line H7 was seeded onto a 24 wellprepared plate in hES medium (1 ml/well). Forty-eight h later, 3 wellsof hES cells were detached using 0.05% trypsin/5 mM EDTA (Sigma) at 37°C., resuspended in 500 μL NHG190 medium, and counted. Stock ofretrovirus construct pGRN354 was thawed on ice immediately prior to use.Growth medium was aspirated from the wells and replaced with 400 μL hESmedium plus 8 μL retrovirus (MOI of 10) and 4 μL of 8 mg/mL polybrenesolution (Sigma). Two h later, 800 μL hES growth medium were added perwell. Each transduced well was refed with 1 mL fresh hES medium every 24h.

Four days after transduction, medium was replaced with 1 mL hES growthmedium containing 200 μg/mL geneticin. After 3 days of geneticinselection, the cells were detached with collagenase, triturated,resuspended in 3 mL hES medium, reseeded into one well of a 6-well platecoated with gelatin and NHG190 feeders, and refed with hES medium after24 h. The medium was then again replaced with hES medium containinggeneticin and refed every 24 h. Undifferentiated colonies survived theselection, and have been maintained for over 3 months. FACS analysisshowed that 50-65% of the selected cells express GFP, albeit at lowlevels. The karyotype of the cells was normal.

FIG. 3 shows GFP expression of hES cells transduced with retrovirus andthen differentiated. The hES cell line H7 was plated on drug-resistant(NHG190) feeder layers, infected with GRN354 and selected for resistanceto the drug G418. Transduced cells were expanded and maintained underG418 selection for multiple passages. The cells were transferred tosuspension culture to form embryoid bodies, allowed to differentiate for4 days, and then plated in 20% FBS medium for 1 week. After extensivedifferentiation occurred, cultures were fixed in 4% paraformaldehyde andphotographed under fluorescence for GFP expression. Many of thedifferentiated cells express higher levels of GFP than theundifferentiated transfected hES line, consistent with differentialactivation of the MESV-LTR in different cell types.

Example 8 Transfection of Feeder-Free hES Cells

In this example, hES cells maintained in feeder-free culture on lamininin conditioned medium were genetically modified by transfecting with aplasmid carrying green fluorescent protein (GFP) driven by the CMVpromoter.

mEF conditioned medium was prepared as described earlier. mEFs wereirradiated and seeded at about 5.7×10⁴ cells/cm². After at least 16hours the medium was exchanged with hES medium including 4 ng/mL addedhbFGF. Conditioned medium was collected daily for feeding of hEScultures. Before addition to the hES cultures this medium wassupplemented with an additional 4 ng/mL of hbFGF. Where needed forselection of stable transfectants, the mEF-conditioned medium wassupplemented with 200 μg/mL geneticin (Sigma cat. # G5013).

H9 hES cells maintained on mEF feeder layers were harvested fromcultures by incubation with ˜200 units/mL collagenase IV at 37° C. for10 min. Cells were dissociated and resuspended in regular hES culturemedium or mEF-conditioned medium. Cells in the regular medium were thenre-seeded onto mEF feeder layers and cells in the mEF-conditioned mediumwere plated onto Matrigel® or laminin. Seeding density for all cultureswas approximately 4×10⁴ cell/cm². Cells on feeder layers were maintainedin regular medium while cells on matrices were maintained in mEF-conditioned medium for 1 or 2 days before the transfection. Conditionedmedium was replaced every 24 h.

hES cell cultures were transfected with Lipofectamine 2000™ as describedabove. FACS analysis of GFP expression was conducted as follows. hEScells were harvested using 0.5 mM EDTA in PBS and resuspended atapproximately 1×10⁶ cells/test. Cells were washed in a solutioncontaining PBS plus 2% FBS, 0.1% sodium azide, and 2 mM EDTA. SSEA-4staining was performed in the same buffer using antibody obtained fromthe Developmental Studies Hybridoma Bank (University of Iowa, Iowa City)at 1:15 dilution. Isotype matched controls were obtained from Sigma,(St. Louis Mo., USA). Cells were incubated with antibodies in a finalvolume of 100 μl for 30 min at 4° C., washed and incubated with ratanti-mouse K chain antibodies conjugated with PE (Becton Dickinson, SanJose, Calif.) at 4° C. for 30 min. Samples were washed as before andanalyzed for GFP and SSEA-4 expression on FACScalibur™ flow cytometer(Becton Dickinson, San Jose, Calif.) using CellQuest™ software.

hES cells of the H9 line maintained on laminin in mEF-conditioned mediumwere transfected with a plasmid carrying GFP driven by the CMV promoterat 24 or 48 h after plating. Initial experiments used a mixture of 5 μgof plasmid and 12 μL of Lipofectamine 2000™. Cells received 1 mL ofDNA/lipid complex and were incubated for 4 h at 37° before the additionof 3 mL of mEF-conditioned medium, and then monitored for GFP expression24 h after transfection.

FIG. 4 shows the results of this experiment. Panel A: morphology of H9cells maintained on laminin. Panel B: GFP-positive cells observed in thesame colony shown in A. Panel C: FACS analysis of % GFP-positive cellsin SSEA-4 high population(undifferentiated cells). Cells weretransfected 24 (bar 1 and 2) or 48 h (bar 3 and 4) after the seeding andanalyzed 24 (bar 1 and 3) or 48 h (bar 2 and 4) after the transfection.Bright green cells were observed in compact areas of undifferentiated EScolonies on laminin 24 h after transfection (Panels A & B). Transfectionat 48 h after initial seeding gave the highest efficiency: 38% of thecells were GFP-positive as determined by FACS analysis 24 h after thetransfection (Panel C).

The next experiment compared the transfection efficiency of H9 cellsmaintained on Matrigel® or laminin-coated plates in mEF-conditionedmedium with cells maintained on mEF feeders. Cells on feeder layersmaintained in regular medium were used as a control. Morphologicaldifferences between cells on feeders and cells off feeders were observed1 or 2 days after seeding. Colonies on feeders were more compact thancells maintained off feeder layers; individual hES cells in feeder-freecultures were less compact and flatter. There was no significantdifference in cell or colony morphology between cells on laminin andcells on Matrigel. These cells were transfected with a plasmidexpressing GFP driven by the CMV promoter 2 days after seeding.Twenty-four hours after the transfection, cells were examined for GFPexpression under a fluorescence microscope.

The cells were maintained on mEF feeders in regular medium (mEF/RM), onlaminin in medium conditioned by mEF (Laminin/CM) or on Matrigel® in theconditioned medium (Matrigel/CM). Bright green cells were observed inundifferentiated hES colonies of feeder-free cultures. In contrast, veryfew green cells were found in colonies on feeders. FACS analysis showedthat 16% of cells on Matrigel® and 14% of cells on laminin were GFPpositive in SSEA-4 high population while only 5% of cells on feederswere positive. These results indicate that transfection efficiency issignificantly increased by using feeder-free conditions.

The next experiments evaluated the effects of 1) the ratio of DNA:lipid;2) adding the DNA/lipid complex to cells 4 h prior to the addition ofmEF-conditioned medium vs. addition of the complex to cells in thepresence of mEF-conditioned medium; and 3) use of Lipofectamine 2000™vs. FuGENE™.

Transfection using Lipofectamine2000™ is described above. Transfectionwith FuGENE™ was conducted as follows. The plasmid DNA (5-10 μg ofpEGFP-C1, ClonTech cat. # 6084-1) was diluted in water to a final volumeof 100 μl. In pilot experiments, 5-30 μL of FuGENE™ were added tosufficient OptiMEM™ to achieve a final volume of 100 μL. The DNAsolution was then added slowly to the FuGENE™ solution and mixed gently.The mixture was incubated at room temperature for 30 min before beingsupplemented with 800 μl of OptiMEM™. Cells were washed with 3 mL ofpre-warmed OptiMEM™ and incubated in 1 mL of the DNA/lipid mixturesolution at 37° C. for 4 h. In some experiments, after 4 h the wellsreceived an additional 2 mL of mEF-conditioned medium; in others theDNA/lipid mixture was added to wells containing 2 mL of mEF-conditionedmedium and the cells were incubated in this mixture overnight.

Highest efficiencies were obtained under the following conditions: Bar1=a mixture of 5 μg plasmid plus 12 μl of Lipofectamine 2000™, adding 1mL of the DNA/lipid mixture to wells containing 2.5 mL ofmEF-conditioned medium and incubating the cells in this mixtureovernight. Bars 2 & 3=a mixture of 10 μg plasmid plus 15 μl of FuGENE™and incubating the cells in 1 mL of the DNA/lipid mixture for 4 h beforeadding 2.5 mL of mEF-conditioned medium. L=Lipofectamine2000™;F=FuGENE™.

To investigate whether the feeder-free hES cells undergo stable geneticmodification, H1 hES cells maintained on Matrigel® were cotransfectedwith a mixture of 7.5 μg plasmid carrying β-galactosidase driven by theEF1a promoter, and 2.5 μg of plasmid carrying the PGK promoter drivingthe neophosphotransferase gene. The cells were transfected 48 h afterplating them on Matrigel® in mEF-conditioned medium. 10 μg of plasmidplus 15 μl of FUGENE™ were incubated with the cells in 1 mL for 4 hbefore adding 2.5 mL of mEF-conditioned medium. After 48 h, medium wasexchanged for mEF-conditioned medium supplemented with 200 μg/mLgeneticin. Cultures were maintained in this geneticin-containing mediumwith daily medium exchange for over 21 days. All mock-transfectedcultures (i.e., those that received FuGENE™ mixed with water rather thanplasmid) died within 48-72 h. Drug resistant colonies arose in the wellstransfected with both FuGENE™ and plasmid at a frequency of about 1 into 10⁵ originally transfected cells. The colonies were maintained ingeneticin-containing mEF-conditioned medium and expanded.

Example 9 Preparation of Vectors in Which a Thymidine Kinase Gene isUnder Control of an hTERT Promoter Sequence

The lambda clone designated λGΦ5 containing the hTERT promoter isdeposited with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110 U.S.A., under Accession No. 98505.λGΦ5 contains a 15.3 kbp insert including approximately 13,500 basesupstream from the hTERT coding sequence.

A Not1 fragment containing the hTERT promoter sequences was subclonedinto the Not1 site of pUC derived plasmid, which was designated pGRN142.A subclone (plasmid “pGRN140”) containing a 9 kb Ncol fragment (withhTERT gene sequence and about 4 to 5 kb of lambda vector sequence) waspartially sequenced to determine the orientation of the insert. pGRN140was digested using SalI to remove lambda vector sequences, the resultingplasmid (with removed lambda sequences) designated pGRN144. The pGRN144insert was then sequenced.

SEQ. ID NO:1 is a listing of the sequence data obtained. Nucleotides1-43 and 15376-15416 are plasmid sequence. Thus, the genomic insertbegins at residue 44 and ends at residue 15375. The beginning of thecloned eDNA fragment corresponds to residue 13480. There are Alusequence elements located ˜1700 base pairs upstream. The sequence of thehTERT Insert of pGRN142 can now be obtained from the GenBank websiteunder Accession PGRN142.INS AF121948. Numbering of hTERT residues forplasmids in the following description begins from the translationinitiation codon, according to standard practice in the field. The hTERTATG codon (the translation initiation site) begins at residue 13545 ofSEQ. ID NO:1. Thus, position −1, the first upstream residue, correspondsto nucleotide 13544 in SEQ. ID NO:1.

Expression studies were conducted with reporter constructs comprisingvarious hTERT upstream and intron sequences. A BgIII-Eco47III fragmentfrom pGRN144 (described above) was digested and cloned into theBgIII-NruI site of pSEAP2Basic (ClonTech, San Diego, Calif.) to produceplasmid designated pGRN148. A second reporter-promoter, plasmid pGRN150was made by inserting the BgIII-FspI fragment from pGRN144 into theBgIII-NruI sites of pSEAP2. Plasmid pGRN173 was constructed by using theEcoRV-Stul (from +445 to −2482) fragment from pGRN144. This makes apromoter reporter plasmid that contains the promoter region of hTERTfrom approximately 2.5 kb upstream from the start of the hTERT openreading frame to just after the first intron within the coding region,with the initiating Met codon of the hTERT open reading frame changed toLeu. Plasmid pGRN175 was made by APA1(Klenow blunt)-SRF1 digestion andreligation of pGRN150 to delete most of the Genomic sequence upstream ofhTERT. This makes a promoter/reporter plasmid that uses 204 nucleotidesof hTERT upstream sequences (from position −36 to −117). Plasmid pGRN176was made by PML1-SRF1 religation of pGRN150 to delete most of the hTERTupstream sequences. This makes a promoter/reporter plasmid that uses 204nucleotides of hTERT upstream sequences (from position −36 to −239).

Levels of secreted placental alkaline phosphatase (SEAP) activity weredetected using the chemiluminescent substrate CSPD™ (ClonTech). SEAPactivity detected in the culture medium was found to be directlyproportional to changes in intracellular concentrations of SEAP mRNA.The pGRN148 and pGRN150 plasmids (hTERT promoter-reporter) and thepSEAP2 plasmid (positive control, containing the SV40 early promoter andenhancer) were transfected into test cell lines. pGRN148 and pGRN150constructs drove SEAP expression as efficiently as the pSEAP2 inimmortal (tumor-derived) cell lines. Only the pSEAP2 control gavedetectable activity in mortal cells.

The ability of the hTERT promoter to specifically drive the expressionof the thymidine kinase (tk) gene in tumor cells was tested using avariety of constructs: One construct, designated pGRN266, contains anEcoRI-FseI PCR fragment with the tk gene cloned into the EcoRI-FseIsites of pGRN263. pGRN263, containing approximately 2.5 kb of hTERTpromoter sequence, is similar to pGRN150, but contains a neomycin geneas selection marker. pGRN267 contains an EcoRI-FseI PCR fragment withthe tk gene cloned into the EcoRI-FseI sites of pGRN264. pGRN264,containing approximately 210 bp of hTERT promoter sequence, is similarto pGRN176, but contains a neomycin gene as selection marker. pGRN268contains an EcoRI-XbaI PCR fragment with the tk gene cloned into theEcoRI-XbaI (unmethylated) sites of pGRN265. pGRN265, containingapproximately 90 bp of hTERT promoter sequence, is similar to pGRN175,but contains a neomycin gene as selection marker.

These hTERT promoter/tk constructs, pGRN266, pGRN267 and pGRN268, werere-introduced into mammalian cells and tk/+stable clones (and/or masspopulations) were selected. Ganciclovir treatment in vitro of the tk/+cells resulted in selective destruction of all tumor lines tested,including 143B, 293, HT1080, Bxpc-3′, DAOY and NIH3T3. Ganciclovirtreatment had no effect on normal BJ cells.

FIG. 5 is a map of the TPAC adenovector pGRN376. It was made by cloningthe NOT1-BAMH1 fragment from pGRN267 into the NOT1-BGL2 sites of pAdBN(Quantum Biotech). The 7185 bp vector comprises the herpes simplexthymidine kinase (TK) gene under control of the medium-length hTERTpromoter sequence.

Example 10 Transduction of hES Cells with a Thymidine Kinase Construct

These experiments test the effect of the pGRN376 vector described in thepreceding Example on hES cells. The vector contains the herpes virusthymidine kinase gene under control of the telomerase reversetranscriptase promoter. Expression of the thymidine kinase gene in cellsshould render them susceptible to toxicity from the prodrug ganciclovir.

Undifferentiated H1 cells were plated into 24 well plates (1 confluentwell of a 6 well plate split into 24 wells of a 24 well plate). After 48h, some wells were infected with the TPAC vector at an MOI of 30 or 100.Four h after addition of the viral vector, medium was exchanged for newmouse embryonic fibroblast conditioned medium (mEF-CM); some wellsreceived medium supplemented with 30 μM ganciclovir (GCV). Cells exposedto GCV were re-fed with mEF-CM containing 30 μM GCV daily for 4 days. Ondays 2,3, and 4 after the initiation of GCV treatment, wells wereharvested and analyzed by flow cytometry to assess changes in 1) totalcell number and 2) cell viability (measured by PI exclusion).

FIG. 6 shows the results of this experiment. No change in total cellnumber was detected at MOI of 30 in the absence of GCV; but there wassome decrease at MOI of 100 in absence of GCV starting at 48 h. Evidencefor toxicity of GCV alone was detected: wells receiving GCV alonecontained approximately 55% as many cells as the control wells on day 2,diminishing to 40% by day 4. Wells receiving GRN376 at MOIs of 30 or 100cultured in the presence of GCV showed identical results: by day 2,these wells contained 18% of the cells contained in the control wells,while at days 3 and 4 these wells contained 6% and 8% of the cells inthe control wells.

Slight toxicity was seen at MOI of 100 at day 4 in the absence of GCV(50% cells in ES gate vs. 83% for the control cells). Some toxicity ofGCV alone was observed at d2, 75% cells in ES gate (vs. 85% control); atday 3, 68% (vs. 82% control); at day 4, 50% (vs. 65% control). Wellsreceiving GRN376 at MOIs of 30 or 100 cultured in the presence of GCVshowed similar results: by day 2, these wells contained 24-28% cells inthe ES gate, at day 3 they contained 19-22% cells in the ES gate, and atd4 these wells contained 12% cells in the ES gate. Thus, GRN376 plus GCVis effective at killing undifferentiated hES cells at an MOI as low as30.

Titration Experiment

Undifferentiated Hi cells were plated into 24 well plates (1 confluentwell of a 6 well plate split into 24 wells of a 24 well plate). After 48h, some wells were infected with pGRN376 at an MOI of 30. Four h afteraddition of the viral vector, medium was exchanged for new mEF-CM; somewells received medium supplemented with 5, 10, 20, 30, or 40 μMganciclovir (GCV). Cells exposed to GCV were re-fed with mEF-CMcontaining GCV daily for 2 days. On day 2 after the initiation of GCVtreatment, wells were harvested and analyzed by flow cytometry to assesschanges in total cell number, and cell viability (measured by PIexclusion).

FIG. 7 shows the results of this experiment. ˜20 μM GCV was optimalunder the conditions tested.

Comparison of Different hES Lines

Undifferentiated hES of lines designated Hi and H7 cells were platedinto 24 well plates (1 confluent well of a 6 well plate split into 24wells of a 24 well plate). After 48 h, some wells were infected withpGRN376 at an MOI of 30. Four h after addition of the viral vector,medium was exchanged for new mEF-CM; some wells received mediumsupplemented with 20 μM GCV. Cells exposed to GCV were re-fed withmEF-CM containing GCV daily for 3 days. On day 4 after the initiation ofGCV treatment, wells were harvested and analyzed by flow cytometry toassess changes in total cell number.

FIG. 8 shows the results. The total cell number demonstrated decreasesin cell number for both lines after TPAC vector treatment. Thus,different hES cell lines respond to the TPAC vector. In subsequentstudies, the H9 cell line was also found to be highly sensitive to GCVafter TPAC vector treatment.

Example 11 Selection of Differentiated Cells

In this experiment, hES cells were treated with retinoic acid (RA) ordimethyl sulfoxide (DMSO), and then analyzed for hTERT and OCT-4expression after treating with TPAC.

Undifferentiated H1 cells were plated into 24 well plates (1 confluentwell of a 6 well plate split into 24 wells of a 24 well plate). 24 hlater, some wells were re-fed with mEF-CM containing either 500 nM RA or0.5% DMSO; wells were re-fed with medium supplemented with RA or DMSOfor the remainder of the experiment. After 7 days of treatment with RAor DMSO, cells were infected with GRN376 at an MOI of 30.

Four h after addition of the viral vector, medium was exchanged for newmEF-CM (plus RA or DMSO where appropriate); some wells also receivedmedium supplemented with 20 μM ganciclovir (GCV). Cells exposed to GCVwere re-fed with mEF-CM containing GCV daily for 3 days. On day 3 afterthe initiation of GCV treatment, wells were harvested and analyzed byflow cytometry to assess changes in total cell number. Additional wellswere used in an effort to culture out any remaining undifferentiatedstem cells; the medium of these wells was changed to mEF-CM (without RA,DMSO, or GCV). Cells were refed with mEF-CM every day for 7 days, thenharvested for isolation of RNA. These samples were analyzed byquantitative RT-PCR for the expression of hTERT and OCT-4.

FIG. 9 shows that the cell number decreased after TPAc treatment. After7 days of drug pretreatment followed by TPAC plus GCV, all wellscontained similar cell numbers. During the attempt to culture outsurviving stem cells, the wells became confluent with highlydifferentiated appearing cells; no undifferentiated hES cells wereobvious. Wells containing cells that had been pre-treated with RA weredistinct in appearance from the cells either pre-treated withunadulterated mEF-CM or treated with mEF-CM plus DMSO. RT-PCR analysis(Lower Panel, non-quantitative, 35 cycles) showed that the survivingcells from the mEF-CM or DMSO-treated wells had no detectable OCT-4expression, while 2 out of 4 RA-pre-treated samples presented very weakOCT-4 PCR products.

Thus, no detectable undifferentiated cells survive TPAC treatmentfollowed by subsequent culture of wells grown in mEF-CM or mEF-CM plusDMSO. RA pre-treatment leads to detection of low levels of OCT-4 in thesurviving cells. It is not clear whether this reflects persistence ofundifferentiated stem cells or induction of another cell type thatexpresses OCT-4.

Example 12 Chimeric α(1,3)galactosyltransferase for Optimal XenoantigenExpression

It is a hypothesis of this invention that elimination ofundifferentiated cells will be enhanced and have greater efficiency witha higher density of xenodeterminant. When α(1,2)fucosyltransferase andα(1,3)galactosyltransferase are coexpressed, the α1,2FT productdominates (U.S. Pat. No. 6,166,288). This is because the cytoplasmicdomains of the two transferase enzymes direct them to different regionsof the Golgi (Osman et al., supra). α1,2FT is naturally expressed invirtually all metabolically active human cells.

To overcome this problem, a chimeric protein was designed in which anon-human α1,3GT catalytic domain was joined to the human α1,2FTcytoplasmic domain—thus producing a protein that should position itselfin the Golgi in a more advantageous position.

The mouse α1,3GT coding sequence was isolated from mouse kidney totalRNA by RT/PCR using primers 5′ of the start codon and 3′ of the stopcodon (5′- ggcctgtac tacatttgcctgga -3′, SEQ. ID NO: 14; 5′-gaaatagtgtcaagtttccatcacaa -3′, SEQ. ID NO:15). The PCR product was subcloned intopGEM T Easy™ (Promega) and used as a template for a PCR reaction toreplace the cytoplasmic tail with a α1,2FT cytoplasmic tail. The same 3′primer was used with an internal 5′ primer containing the α1,2FT codons(underlined) to replace the mouse α1,3GT cytoplasmic tail(5′-cgatgtggctgcg gagccaccggcag gtaatcctgttg atgctgattgtctc aac -3′,SEQ. ID NO:16). The resulting PCR product was subcloned into pGEM TEasy™ (Promega).

MITMLQDLHVNKISMSRSKSETSLPSSRSGSQEKIMNVKGKVILLMLIVS    A (SEQ. ID NO:17)                                 MWLRSHRQVILLMLIVS    B (SEQ. ID NO:18)                                 MWLRSHRQVVLSMLLVS    C (SEQ. ID NO:19)                                   MNVKGRVVLSMLLVS    D (SEQ. ID NO:20)A: mouse α1,3GT N terminal sequence B: constructed chimeric mouse α1,3GTN terminal sequence (α1,2FT underlined) C: chimeric porcine α1,3GT Nterminal sequence D: porcine α1,3GT N terminal sequence

-   -   chimeric galT    -   1121 bp

Example 13 α(1.3)galactosyltransferase under Control of the EndogenoushTERT Promoter

Human ES cells are engineered to express α(1,3)galactosyltransferase(α1,3GT) in several ways. The general strategy is illustrated in FIG.10.

The first option is a two-step targeting approach. A promoter traptargeting construct is generated to introduce a promoterless neo flankedby incompatible lox sites into intron 2 of the hTERT gene. The lox-neocassette also contains a splice acceptor site adjacent to the 5′loxPsite. In this way, an mRNA is transcribed which encodes a fusion ofneomycin phosphotransferase with the first few amino acids of thetelomerase catalytic subunit to render targeted clones resistant toselection in G-418. The great majority of random integrants will notproduce a transcript for G-418 resistance unless fortuitously integratedinto an intron of an expressed gene. In a second step, the floxed neo isreplaced with the ovine (α1,3GT encoding sequence by recombinationmediated cassette exchange (RMCE) using a cre expressing plasmid (Lee etal., Gene 216 55, 1998; Kolb et al., Gene 227:21, 1999). Using themutated loxM site eliminates recombination events in which neo isremoved without replacement by α1,3GT and ensures directionality of theinsertion. Between the α1,3GT and the 3′loxM site is thetetracyclin-inducible promoter TetO7-CMVm and a fusion of the codingregions of exons 1 and 2 of the hTERT gene, such that second steptargeting will (in the presence of transactivator) render the completehTERT open reading frame inducible in tetracyclin or doxycycline (dox).In a subsequent step, targeted clones are transfected with thetransactivator rtTAVB, which binds the tet operator in TetO7-CMVm andinduces transcription only in the presence of dox (Rossi et al. NatureGenetics 20 389, 1998).

In single step targeting experiments, neo is introduced with an internalribosomal entry site (IRES) 3′ to the α1,3GT coding region. In thisinstance, the α1,3GT encoding sequence is truncated before thepolyadenylation signal and is transcribed directly from the hTERTpromoter. The IRES allows transcription of a bicistronic message fromwhich both proteins are translated. This single-step approach has theadvantage that the initial modification is directly useful without asecond cloning step. The two-step procedure, on the other hand, offersthe advantage that a variety of open reading frames can be introduced toa single clone through cre-mediated recombination.

Three features of α1,3GT-expressing hES cells are desirable: 1. That allundifferentiated hES cells are sensitive to human serum; 2. That alldifferentiated hES derivatives are resistant to human serum; 3. That intargeted clones, telomerization can be induced in vitro leading toincreased proliferative life span.

Expression of α1,3GT in modified hES cells is monitored by exposure ofdifferentiated and undifferentiated cultures to fresh human serum(containing natural antibody to the Galα(1,3)Gal epitope andcomplement), and by RT-PCR. With appropriate levels of expression, thereshould be complement mediated lysis of undifferentiated cells, but noeffect on differentiated cells. Based upon the frequency of randommutation at the HPRT locus in murine ES cells, a reversion frequency ofapproximately 10-7 can be expected. Hence, a similar backgroundfrequency among undifferentiated hES cells exposed to human serumindicates that cells containing the α1,3GT transgene are effectivelydestroyed. Loss of α1,3GT in surviving cells can be confirmed bySouthern analysis. Thus, hES derived cells can be exposed to human serumin vitro immediately prior to transplantation, and selection againstundifferentiated cells will continue to be active in vivo.

Clones in which the targeted hTERT allele is controlled by theTetO7-CMVm promoter are transfected with the transactivator rtTAVB. Upto 10 rtTAVB expressing clones derived from previously targetedsubclones are assessed for responsiveness to induction and for telomerelength. Expression and function of hTERT is monitored in vitro andcompared with and without dox induction of the modified hTERT locus byRT-PCR and by TRAP assay, respectively.

References

-   1. Insertion of a foreign gene into the β-casein locus by    Cre-mediated site-specific recombination. Kolb, A., Ansell, R.,    McWhir J., Siddell, S. Gene Vol. 227:21-31, 1999.-   2. Viable offspring derived from fetal and adult mammalian cells.    Wilmut I, Schnieke A E, McWhir J, Kind A J, Campbell K H S. Nature    385:10-813,1997.-   3. Selective ablation of differentiated cells permits isolation of    embryonic stem cell lines from murine embryos with a non-permissive    genetic background. McWhir J, Schieke A E, Ansell R, Wallace H,    Colman A, Scott A R, Kind A J. Nature Genetics, 14:223-226, 1996.-   4. Sheep cloned by nuclear transfer from a cultured-cell line.    Campbell K H S, McWhir J, Ritchie W A, Wilmut I. Nature, 380:64-66,    1996.-   5. Use of double-replacement gene targeting to replace the murine    alpha-lactalbumin gene with its human counterpart in embryonic    stem-cells and mice. Stacey A, Schnieke A, McWhir J, Cooper J,    Colman A, Melton D W. Mol. Cell Biol. 14:1009-1016, 1994.-   6. Mice with DNA-repair gene (ercc-1) deficiency have elevated    levels of p53, liver nuclear abnormalities and die before weaning.    McWhir J, Selfridge J, Harrison D J, Squires S, Melton D W. Nature    Genetics 5:217-224, 1993.-   7. In-vivo analysis of pim-1 deficiency. Laird P W, Vanderlugt N M    T, Clarke A, Domen J, Linders K, McWhir J, Bems A, and Hooper M.    Nucleic Acids Res. 21:4750-4755, 1993.-   8. Gene targeting using a mouse hprt minigene hprt-deficient    embryonic stem-cell system—inactivation of the mouse ercc-1 gene.    Selfridge J, Pow A M, McWhir J, Magin T M, Melton D W. Somatic Cell    Mol. Genet. 18:325-336, 1992.-   9. Switching amino-terminal cytoplasmic domains of    alpha(1,2)fucosyltransferase and alpha(1,3)galactosyltransferase    alters the expression of H substance and Gal alpha(1,3)Gal.    Osman, N. et al., J. Biol. Chem. 271:33105, 1996.

It will be recognized that the compositions and procedures provided inthe description can be effectively modified by those skilled in the artwithout departing from the spirit of the invention embodied in theclaims that follow.

1. A method of producing differentiated cells, comprising a) obtaining acell population comprising undifferentiated stem cells that have beengenetically altered to contain a nucleic acid molecule comprising P-X,wherein X is nucleic acid sequence that causes expression of a cellsurface antigen not normally expressed in the population, and P is atranscriptional control element operatively linked to X, such that thesurface antigen is expressed in undifferentiated cells; b) causing atleast some undifferentiated cells in the population to differentiate;and c) culturing the remaining differentiated cells.
 2. The method ofclaim 1, wherein the undifferentiated stem cells are primate pluripotentstem (pPS) calls.
 3. The method of claim 1, wherein X encodes aglycosyltransferase.
 4. The method of claim 3, wherein X encodes anα(1.3)galactosyltransferase.
 5. The method of claim 3, wherein X encodesan A or B transferase from the ABO Blood Group system.
 6. The method ofclaim 1, wherein P is an OCT-4 promoter or a promoter of telomerasereverse transcriptase (TERT).
 7. The method of claim 1, wherein thecells have been genetically altered using a vector comprising P-X. 8.The method of claim 1, wherein the cells have been genetically alteredto place X Under control of a promoter (P) present in the cell genome.9. The method of claim 1, further comprising depleting undifferentiatedcells from the population by combining the cells the with a ligandspecific for the antigen.
 10. The method of claim 9, wherein the ligandis an antibody or a lectin.
 11. The method of claim 9, comprisingcombining the cells with ligand specific for the antigen, and separatingcells that have not bound the ligand.
 12. The method of claim 9,comprising combining the cell population or progeny thereof withcomplement and antibody specific for the antigen under conditions thatpermit the complement to lyse cells to which the antibody has bound. 13.The method at claim 9, wherein a) comprise genetically altering the cellpopulation such that P-X is transiently expressed in undifferentiatedcells in the population.
 14. The method of claim 13, wherein a)comprises genetically altering the cell population with an adenovirusvector comprising P-X.
 15. The method of claim 9, wherein a) comprisesgenetically altering the cell population such that P-X is inherited byprogeny of cells in the population, and thereby expressed inundifferentiated progeny.
 16. The method of claim 15, wherein a)comprises genetically altering the cell population with a DNA plasmid orretrovirus vector comprising P-X.
 17. A method of depletingundifferentiated stem cells from a cell population, comprising: a)obtaining a cell population that comprises both differentiated cells andundifferentiated stem cells; b) genetically altering undifferentiatedstem cells in the population so that they contain a nucleic acidmolecule comprising P-X, wherein X is nucleic acid sequence that causesexpression of a cell surface antigen not normally expressed in thepopulation, and P is a transcriptional control element operativelylinked to X, such that the surface antigen is expressed in theundifferentiated stem cells; c) depleting undifferentiated cells fromthe population by combining the cells with a ligand specific for theantigen; and d) culturing the remaining differentiated cells.
 18. Themethod of claim 17, wherein the ligand is an antibody or a lectin. 19.The method of claim 17, comprising combining the cells with ligandspecific for the antigen, and separating cells that have not bound theligand.
 20. The method of claim 17, comprising combining the cellpopulation or progeny thereof with complement and antibody specific forthe antigen under condition that permit the complement to lyse cells towhich the antibody has bound.
 21. The method of claim 17, wherein Xencodes a glycosyltransferase.
 22. The method of claim 17, wherein P isa TERT promoter.
 23. The method of claim 17, wherein the promoter is anOCT-4 promoter.
 24. A method for preparing cells, comprising: a)obtaining human embryonic stem (hES) cells that have been geneticallyaltered so as to transcribe a nucleic acid sequence under control of apromoter that preferentially drives transcription in undifferentiatedhES calls, wherein transcription of the nucleic acid causes expressionof a surface antigen not normally expressed by undifferentiated hEScell; b) differentiating the hES cells; and then c) formulating thecells from step b) for administration to a mammalian host.
 25. Themethod of claim 24, wherein the hES cells have been genetically alteredto place the nucleic acid sequence under control of a promoter presentin the cell genome.
 26. The method of claim 24, wherein the hES cellshave been genetically altered to place the nucleic acid sequence undercontrol of a heterologous promoter.
 27. The method of claim 24, whereinthe promoter is a TERT promoter.
 28. The method of claim 24, wherein thepromoter is an OCT-4 promoter.
 29. The method of claim 24, wherein thenucleic acid sequence encodes a cell surface protein not normallyexpressed in human cells.
 30. The method of claim 24, wherein thenucleic acid encodes a glycosyltransferase that causes expression of acell surface carbohydrate to which some humans have naturally occurringantibody.
 31. The method of claim 24, wherein b) comprisesdifferentiating the hES cells into neural cells.
 32. The method of claim24, wherein b) comprises differentiating the hES cells into hepatocytes.33. The method of claim 24, further comprising depletingundifferentiated hES cells before the differentiated cells areformulated for administration to a mammalian host.
 34. The method ofclaim 33, wherein the depleting step comprises adding a ligand specificfor the antigen, and separating cells that have not bound the ligand.35. The method of claim 33, wherein the depleting step comprises addingcomplement and antibody specific for the antigen under conditions thatpermit the complement to lyse cells to which the antibody has bound. 36.A method of depicting undifferentiated stem cells from a mixed cellpopulation, comprising: a) obtaining a mixed cell population thatcomprises both differentiated cells and undifferentiated human embryonicstem (hES) calls; b) genetically altering the hES cells in the mixedcell population so as to transcribe a nucleic acid sequence undercontrol of a promoter that preferentially drives transcription inundifferentiated hES cells, wherein transcription of the nucleic acidcauses expression of a surface antigen not normally expressed byundifferentiated hES cells, thereby producing a genetically altered cellpopulation; c) depleting undifferentiated cells from the geneticallyaltered cell population by using a lectin or antibody specific for theantigen, thereby producing a more homogeneous cell population; and d)formulating the more homogeneous cell population for administration to amammalian host.
 37. The method of claim 36, wherein the promoter is aTERT promoter.
 38. The method of claim 36, wherein the promoter is anOCT-4 promoter.
 39. The method of claim 36, wherein c) comprisescombining the genetically altered cell population with a lectin orantibody specific for the antigen, and separating cells that have notbound the lectin or antibody.
 40. The method of claim 36, wherein c)comprises combining the genetically altered cell population withcomplement and antibody specific for the antigen under conditions thatpermit the complement to lyse cells to which the antibody has bound.